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This is to certify that the

dissertation entitled
DEVELOPMENT OF SUGARBEEI‘ TISSUE CULTURE SYSTEMS: SOMATIC

EMERYOGENESIS, AND EVALUATICN OF ALTERNATIVE NITRWEN SOURCES

presented by

Chia-Jung Tsai

has been accepted towards fulfillment
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MSU 10 An Affirmative Halon/Emmi Opponunlty 1m

DEVELOPMENT OF SUGARBEET TISSUE CULTURE SYSTEMS :
SOMATIC EMBRYOGENESIS, AND EVALUATION OF ALTERNATIVE
NITROGEN SOURCES

By

Chia-Jung Tsai

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Sciences

1997

ABSTRACT

DEVELOPMENT OF SUGARBEET TISSUE CULTURE SYSTEMS :
SOMATIC EMBRYOGENESIS, AND EVALUATION OF ALTERNATIVE
NITROGEN SOURCES

By
Chia-Jung Tsai

SomaficanbryogmesisasweflassolemuOgmeumforshMmMJeufdisc
W,mdmspmsimcMmrephfingswememmmedformepumoscoffinflierdcvdopmg
sugarbeet '(Beta vulgaris L.) tissue culture systans. Somatic embryos could be used for
pmagafiomforaflficiflsewsmrforgmemsfirpWEademMmemodsbrmifiafim
anddevelopmcutcouldbedevised. Twogcuotypes, REL-1 andREL-2,wereusedtomeasurethe
level of embryo production from suspensions plated on MS (Mumshige and Skoog) medium
containing combinations of abscisic acid (ABA) with difi‘ereut concentrations ofotber growth
regulators, sucrose, or difl‘erent sole N sources. Abscisic acid at 0.1-0.3 myL consistently
hupmvedanbryoproducfimhaflammimafis,andsfimflatedstproducfimaswcH.A
combination ofNAA (0.3 mg/L) and ABA (0.1 mg/L) gavethe highestembryo yield (77 embryos
permlofsuspeusionplatedout). RELZwasamuchmoreprolificembryoandshootproduccr
thanREL-l.Themgeofanbryopmducfionwasmorethanthirty-fivefoldbetmthctwo
gmotypes,whereasflwmgeofyowflimgulaorefie08wasmgwammM-fom.1hmems
mdifiumwmfliewnvemimmeofanbrymimownmldephnflcmamdingmdiflerunlmgm
categories (0.5-1.9; 2.0-2.9; 3.0-3.9 mm) in either REL-l (78, 81, and 85%, respectively) or REL-
2 (87, 89, and 87%, respectively).

lnordertoinifiatedevelopmernofasystemformaldngarfificial seeds, isolatedsomaticanbryos
wemmcapSMacd.WimRBI»2,memcepSMafimofanbryosvufl12%sodiumalgimmMS

medimnorinHzoresultedin8l‘yo,and64%conversion,respccfively,whenplacedonmmiem

agarplates.Afleré4daysofstomgeat4°€,theconversimmteofembryosmcapsulatedwith
alginate in MS medium or in H20 fell to 45% and 20%, respectively.
lnordertoidentifymcdiaforcellselectionofbiochemicalmutantsoroftransgcnicsthatmigln
have reduced levels of nitrogenous pmwssing impurities or to identify superior media for
developmental responses, the ability ofeight endogenous N-containing compounds to serve as sole
Nsource(15,30,60,and/or90mM)forsuspensionplafingcaflusgmwth,shootculhire,aswefl
aslcafdisccultureofclone REL-l wasevaluated. Nitrate (N03), ammonium (NI-I4),glutamine
(GLN), glutamate (GLU), andmassoleNsomces supported plating callus growth. N03,NI-L,
urea, andGLN supported shoot multiplication and flesh weight increase of Shoot cultures. N03,
NI-L, urca, GLN, and proline (PRO) supported callus initiation from leaf discs; N03, urea, GLN,
mPROfinfiamppofledshodregmemfimfiommedisccaflusmmnparedwtheMSNnnxof
N03(20mM)andNI-L(40mM).Glycinebetaine(BEl')andcholine(CHO)didnotsupportgrowth
inanysystemPlafingsoneiflierNogorNTLassoleNsoumedidnotdifi‘erinsaisifivitytoflie
mumeupmlmmenmphmylglyoxflQGO),mggesfingmaPGOhcksmespxifidtyforuxm
selectionfornmtantsofnitrateuptake.'I‘heabilityofGLNorGLUtoserveastthsmrcemay
prxludemeuuxformflxlecfimofgmeficvadamawmulafinglessofflmepmmg
impurities. Moreover, mutants or transgenics able to utilize BET might be selectable at media

containingBETasaNsource.

Dedicated
to my parents

iv

ACKNOWLEDGMENTS
I would like to extend my sincere appreciation to my major professor Dr. Joseph W.
Saunders, for his patient instruction, constant encouragement, and valuable advice. I am
particularly thankful to the members of my committee: Dr. Frank G. Dennis, Dr. Mariam
B. Sticklen and Dr. Joanne H. Whallon for their valuable suggestions and contributions to

my graduate experience. Finally, special and cordial thanks to my parents and my husband

for their concern, support and encouragement.

TABLE OF CONTENTS

Page
LIST OF TABLES ........................................................................... vi
LIST OF FIGURES .......................................................................... vii
LITERATURE REVIEW ................................................................... l
Adventitious shoot regeneration ......................................................... 2
Multiplication of axillary shoots ......................................................... 4
Shoot regeneration from callus or suspension cultures ............................... 5
Miscellaneous use of callus or suspension cultures .................................... 9
Somatic embryogenesis .................................................................. 10
Ovule culture ......................... ‘ ..................................................... 11
Author culture ............................................................................. 13
Protoplasts and somatic hybridization .................................................. 13‘
Somaclonal variation ..................................................................... 15
Somatic cell selection ..................................................................... 16
Genetic transformation .................................................................... 18
References ................................................ L ................................. 22
CHAPTER 1: SOMATIC EMBRYOS FROM CALLUS OF SUGARBEET
BIOTECHNOLOGY CLONE REL-l ................................ 31
ABSTRACT ............................................................................... 31
INTRODUCTION ........................................................................ 32
MATERIALS AND METHODS ....................................................... 34
Plant materials ........................................................................ 34
Culture media ......................................................................... 34
Initiation of callus and maintenance of cell suspensions ......................... 34
Induction of somatic embryos ....................................................... 35
Germination and conversion of somatic embryos ................................. 35
Scanning electron microscopy ...................................................... 35
Encapsulation of somatic embryos .................................................. 36
Data analysis ........................................................................... 36
RESULTS AND DISCUSSION ......................................................... 36
Induction of somatic embryos ........................................................ 36
Development of somatic embryos ................................................... 37
Conversion of somatic embryos into plantlets ..................................... 37
Scanning electron microscopy ....................................................... 38
Encapsulation of somatic embryos .................................................. 39
REFERENCES ............................................................................. 40

vi

CHAPTER 2: MEDIA AND GENOTYPE EFFECTS ON SOMATIC

EMBRYOGENESIS FROM SUGARBEET CALLUS ............ 56
ABSTRACT ............................................................................... 56
INTRODUCTION ........................................................................ 57
MATERIALS AND METHODS ........................................................ 60

Plant materials ......................................................................... 60
Culture media .......................................................................... 60
RESULTS AND DISCUSSION ......................................................... 63
Initiation of leaf disc callus and suspension cultures .............................. 63
Induction of somatic embryos with ABA and NAA .............................. 63
Induction of somatic embryos with ABA and 2,4-D .............................. 65
Induction of somatic embryos and shoots with ABA and BA ................... 65
Induction of somatic embryos and shoots with ABA and TIBA in the presence
of 1.0 mg/L BA ........................................................................ 67
Induction of somatic embryos with ABA and sucrose ............................ 67
Induction of somatic embryos with ABA and sole nitrogen sources ............ 68
Development of somatic embryos ................................................... 69
REFERENCES ............................................................................. 71

CHAPTER 3 : ENCAPSULATTON, GERMINATTON AND CONVERSION
OF SOMATIC ENIBRYOS IN SUGARBEET CLONE REL-2.96

ABSTRACT ................................................................................ 96
INTRODUCTION ........................................................................ 98
MATERIALS AND METHODS ........................................................ 100
Induction of somatic embryos ....................................................... 100
Germination and conversion of somatic embryos ................................. 101
Encapsulation of somatic embryos .................................................. 101
RESULTS AND DISCUSSION ........................................................ 102
Induction of somatic embryos ....................................................... 102
Encapsulation of somatic embryos .................................................. 103
REFERENCES ............................................................................ 107

CHAPTER 4: EVALUATION OF ALTERNATIVE NITROGEN SOURCES
FOR SUGARBEET SUSPENSION CULTURE PLATINGS IN

DEVELOPNIENT OF CELL SELECTION SCHEMES ........... 121
ABSTRACT ................................................................................ 121
INTRODUCTION ......................................................................... 122
MATERIALS AND METHODS ........................................................ 124
RESULTS .................................................................................. 125
DISCUSSION ............................................................................. 126
REFERENCES ............................................................................ 130

CHAPTER 5: EVALUATION OF SOLE NITROGEN SOURCES FOR SHOOT
CULTURES AND LEAF DISC CULTURES OF SUGARBEET....135

ABSTRACT ................................................................................... 135
INTRODUCTION ............................................................................ 136
MATERIALS AND METHODS ........................................................... 137
Plant materials ............................................................................ 137
Media ...................................................................................... 137
Shoot culture .............................................................................. 137
Leaf disc culture .......................................................................... 137
RESULTS AND DISCUSSION ............................................................ 139
Shoot culture .............................................................................. 139
Leafdisc culture .......................................................................... 139
REFERENCES ............................................................................... 142

viii

LIST OF TABLES

Table 1-1 : The effect of BA and NAA concentrations on somatic embryogenesis.
One mL ofsuspension cellswas plated onto mediawithvarious
combinations of BA and NAA and scored 68 days after plating
Means marked with same letter are not significantly difl‘erent
accordmgtoLSDwrthP<005 ........................ 44

Table 1-2 : Efl‘ect of embryo length on the proportions of somatic embryos
germinating and converting into complete plantlets ......................... 45

Table 1-3 : Comparison of encapsulated somatic embryos with MS medium and H30,
and efi‘ects on the germination and conversion rate on MS medium

(genotype REL-1) ................................................................ 45

Table 3-1 : Efl‘ect of length category on the proportions of somatic embryos
germinating and converting into complete plantlets m the absence

of encapsulation .................................................................. 1 1 1
Table 3-2 : Germination and conversion rates of nonencapsulated somatic
embryos on the MS medium or the soil without cold storage ............ 111

Table 3-3 : Comparison of germination and conversion rates on MS medium
of somatic embryos alginate-encapsulated with either MS medium
oriLOafieratorageat4°C ................................................... 112

Table 3-4 : Comparison of germination rates in soil of encapsulated somatic

embryoswithcitherMSmediumoerOandefi‘ectsonthegermi-
nationratesaficrstorageat4°C .............................................. 112

Tables-1 :Imfdiscculturewithsinglenitrogensoru'cesafier 106daysof
inoculation ........................................................................ 144

ix

Figure 1-1 :

Figure 1-2 :

Figure 1-3 :

Figure 1-4:
Figure 1-5 :

Figure 1-6:

Figure 1-7 :
Figure 1-8 :
Figure 1-9:
Figure 1-10

Figure 1-11
Figure 1-12

Figure 1-13

Figure 1-14

LIST OF FIGURES

The number ofsomatic embryos per ml ofsuspension plated out onto
media with combinations of ABA, NAA, and TIBA 33 days after plate
out. Means marked with same letter are not significantly difi‘erent

according to LSD with P < 0.05 ............................................... 46
Opaque white somatic embryos (1.5 mm long) on the surface of callus
tissue 22 days after suspension plating ........................................ 47
A 3 mm long tricotyledonary somatic embryo 30 days alter suspension
plateout ........................................................................... 47
Isolated somatic embryos (length: 0.5 - 4 mm) .............................. 48
Somatic embryo-derived complete plantlet, 10 days after being transferred
to hormone-flee MS medium (length: 20 mm) .............................. 43
Somatic embryo development into plantlet, and betalain pigment on hypocotyl
after transfer onto hormone-flee MS medium ............................... 49
SEM of an early torpedo stage somatic embryo ............................. 50
SEM showing a torpedo stage somatic embryo ............................. 50
SEM showing a late cotyledonary stage somatic embryo .................. 51
: SEM showing an embryo with radicle development fi'om one polar... . 51

: SEM showing a torpedo stage of somatic embryo attached to the callus.52
: SEM showing a somatic embryo with cotyledons and a little swollen

hypocotyl ........................................................................ 52
:SEM showinganabnormal embryo withaswollen hypocotyl ........... 53
: SEM showing trichomes only occurred on the cotyledonary surface of

embryo ........................................................................... 53

Figure 1-15 : SEM showing the magnification of partially area from Figure 1-14.
The trichomes appeared on the cotyledons of the somatic embryo ............... 54

Figure 1-16 : SEM showing a somatic embryo with trichomes on the cotyledonary surface. 54

Figure 1-17 : Encapsulation of somatic embryos. The gel bead is about 4-5 mm in diameter. 55

Figure 2-1 : Somatic embryo production on primary leaf disc callus (28 mm) mm’ ' ’afing
on hormone-flee MS medium after 58 days. (REL-2) ............................... 76

Figure 2-2 : Somatic embryos on callus initiated fiom a leaf disc on hormone-flee MS
medium. About a third of such calli have rough fuzzy white embryogenic

tissue. (REL-2) ............................................................................ 75
Figure 2-3 : Leafdisc callus (24mm) fiom MS medium with 1 mg/L BA (REL-1) ............ 77

Figure2-4 : LeafdisccallusflomMS mediumwith l mg/LBA (REL-2 alsoknown
as LTR-41) ................................................................................. 77

Figure 2-5 : Friable callus was transferred to liquid hormone-flee MS medium for
suspension culture. (REL-1) ............................................................. 78

Figure 2-6 : Plate out callus with a low number of somatic embryos (opaque white)
onMS mediumwith0.25 mg/LNAAand 0.1 mg/LABAafier29 days.

(REL-1) .................................................................................... 78

Figure 2-7 : The influence ofNAA and ABA on somatic embryo production with
genotype REL-1 after 29 days. Means marked with same letter are not

significantly difl‘erent according to LSD (P<0.05) .................................... 79

Figure 2-8 : The influence ofNAA and ABA on somatic embryo production with
genotype 6926cm312 afier 33 days ..................................................... 80

Figure2-9:PlateoutcalluswithsomaticembryosonMSmcdimnwith1mg/L
NAA and 0.3 mg/L ABA after 40 days. (REL-2) .................................... 81

Figure 2-10 : The influence of NAA and ABA on somatic embryo production with
genotypeREL—Z after 31 days. Means markedwiththesamelettcrare

not significantly difl‘erent according to LSD (P<0.05) ............................. 82

Figure 2-11 : Plate out callus with somatic embryos on MS medium with 0.1 mg/L
ABA after 31 days. (REL-2) ........................................................... 83

Figure 2-12 : Plate out callus with somatic embryos on MS medium with 0.3 mg/L
ABA afier 31 days. (REL-2) .......................................................... 83

Figure 2-13 :

Figure 2-14 :

Figure 2-15 :

Figure 2-16 :

Figure 2-17 :

Figure 2-18 :

Figure 2-19 :

Figure 2-20 :

Figure 2-21 :

Figure 2-22 :

Figure 2-23 :

The influence of NAA and ABA on combined flesh weight of callus

and somatic embryos with genotype REL-2 after 31 days. Means
markcdwiththesameletterarenot significantlydifl'crentaccording

to LSD (P<0.05) .................................................................. 84

The influence of 2,4-D and ABA on somatic embryo production with
genotype REL-2 after 32 days. Means marked with the same letter
are not significantly difi‘erent according to LSD (P<0.05) ................... 85

The influence of BA and ABA on somatic embryo and shoot production
with genotype REL-l after 33 days. Means marked with the same letter
are not significantly difl‘erent according to LSD (P<0.05) ................... 86

Typical response of REL-1 callus to combinations of (0, 0.1, 0.3, 1)
mg/LBAandng/LABAmediaafier35days ............................. 87

Typical response of REL-1 callus to combinations of 1 mg/L BA and
(0, 0.02, 0.2, 2) mg/L ABA media after 35 days ............................. 37

The influence of BA and ABA on somatic embryo and shoot production
withgenotypeREL-Z afier34 days. Meansmarkedwiththesameletter
are not significamly difl‘erent according to LSD (P< 0.05) ................. 88

Typical response of REL-2 callus to combinations of (0, 0.1, 0.3, 1)
mg/LBAandng/LABAmediaafter35days ............................. 89

The influence of TIBA and ABA at 1 mg/L BA on somatic embryo
and shoot production with genotype REL-l afier 36 days. Means
marked with the same letter are not significantly difl‘erent according

to LSD (P<0.05) ................................................................. 90

The influence of TIBA and ABA at 1 mg/L BA on somatic embryo

and shoot production with genotype REL-2 after 36 days. Means

marked with the same letter are not significantly different according

to LSD (P<0.05) .................................................................. 91

The influence of sucrose concentration and presence or absence of
0.2 mg/L ABA on somatic embryo production with genotype REL-2
after 35 days. Means marked with the same letter are not significame

difl‘ercnt according to LSD (P<0. 05) ........................................... 92

The influence of sole nitrogen source and ABA on somatic embryo

and shoot production with genotype REL-1 after 34 days. Means

marked with the same letter are not significantly difl’erent according

to LSD (P<0.05) .................................................................. 93

xii

Figure 2-24 : The influence of sole nitrogen source and ABA on somatic embryo
and shoot production with genotype REL-2 after 34 days. Means
marked with the same letter are not significantly difl‘erent according

to LSD (P<0.05) ............................................................... 94
Figure 2-25 : Difi‘erent stages of somatic embryos were clearly distinguishable at
the callus surface. (REL-2) ................................................... 95
Figure3-lzLeafdisccallusfi'omMSmediumwithBA1mg/L ........................ 113
Figure 3-2 : Somatic embryogenesis after suspension plate-out on MS medium
with0.3mg/LNAAand0.lmyLABAafier3l days ..................... 113
Figure 3-3 : Somatic embryos (0.5-4 mm) were present at callus surface .............. 114

Figure 3-4 : Somatic embryo (4 mm) directly developed into complete plantlet
on the suspension plate-out medium .......................................... 114

Figure 3-5 : Somatic embryo with bipolar converted into cotyledons and roots
simultaneously, 6 days afler being transferred to hormone-flee MS

medium (length: 14 mm) ....................................................... 115
Figure 3-6 : Somatic embryos develop into complete plantlet, 9 days afier being
transferred to hormone-flee MS medium (length: 19 mm) ................ 115

Figure 3-7 : Secondary embryogenesis were observed fi'om the primary somatic
embryo (8 mm), 14 days after being transferred to hormone—flee

MS medium ....................................................................... 115
Figure 3-8 : Isolated somatic embryos (length: 0.5-4 mm) ................................ 1 15
Figure 3-9 : Encapsulation of somatic embryos with sodium alginate.

Thegelbeadisabout4-5mm .................................................. 117
Figure3-10:Thediameterofeachgelbeadisapprordmately4-Smm ................. 117

Figure3-11:Anencapsulatedembryothatgerminatedafierthethirddayonagar
plate .............................................................................. 1 13

Figure 3-12 : An encapsulated embryo that germinated alter the seventh day on
agarplatewithbetalainpigmentonthehypocotyls ....................... 113

Figure 3-13 : An encapsulated embryo that converted into a plantlet after fourteenth
day on hormone-flee MS agar plate, with betalain pigment on the

hypocotyl(length: 15 mm) .................................. 1 19

xiii

Figure 3-14 : An encapsulated embryo with cotyledons and radicle development
simultaneouslyafieer days ofstorageat4°C (length: 14 mm) ..... , ..... 119

Figure 3-15 : An encapsulated embryo that germinated out (with radicle elongation)
of encapsulated coat after 64 days of cold storage without transfer.
Capsuleisabout4mmindiameter ........................................... 120

Figure 4-1: Fresh weight of plating callus after 30 days on MS medium, MS minus
nitrogen (MS-N), or media with various sole nitrogen sources. The mM
concentration of the sole nitrogen source is the number following the
designation of nitrogen form Means marked with same letter are not

significantly different according to LSD with P<0.05 ........................ 132

Figure 4-2: Ability of GLN, GLU and BET to serve as sole C or C+N sources on
fi’esh weight of plating callus. Means marked with the same letter are not

significantly difl’erent according to LSD with P<0.05 ........................ 133

Figure 4-3: Efl‘cct ofPGO on fresh weight ofplating callus after 35 days on media
with 60 mM N03, NHL or a mixture of these (total N=120 mM). Response
toPGOisdisplayed asthepercentage ofthefreshweightintheabsenceof
PGO for each N regime. Means marked with the same letter are not signifi-

cantly diEercnt according to LSD with P<0.0S ............................... 134

Figure 5-1 : Cumulative flesh weights of shoot cultures growing with single nitrogen
sources afier initial culture (35 days) and subculture (subsequent 26 days
), respectively. Means marked with same letter are not significantly

difl'erent according to LSD (P<0.05) .......................................... 145

Figure 5-2 : Increase in shoot number in shoot cultures with single nitrogen sources
after 2 months. Means marked with same letter are not significantly

difl‘erent according to LSD (P<0.05) .......................................... 146
Figure 5-3 : Final medium pH afier shoot culture with single nitrogen sources after
2 months ........................................................................... 147

Frgum54:Caflusingresponseofaflnnedleafdiscswithsinglemmgensomces.
Means of86 dayvalues markedwithsameletterarenot significantly
difl‘erent according to LSD (P<O. 05) .......................................... 148

Figure 5-5: Number ofshoots per callusing leafdisc on single nitrogen sources
after 106 days. Means marked with same letter are not significantly

difl'erentaccordingto LSD(P<0.05) .......................................... 149

Figure 5-6 : Leafdisc culture with single nitrogen sources after 106 days. For
percentage of disc callusing only, means marked with same letter
are not significantly difi'erent according to LSD (P<0.05) .................. 150

Figure 5-7 : Leaf disc expansion with single nitrogen sources after 15 days. Means
marked with same letter are not significantly difl‘erent according to LSD

(P<0.05) ........................................................................... 1 51

LITERATURE REVIEW

Sugarbeet, a naturally cross-pollinating biennial and herbaceous dicotyledon of the
family Chenopodiaceae, is the most economically important sucrose-producing crop in the
temperate and subtropical regions of the world. It supplies about 40 % of the world
supply of sucrose, an extremely important commodity on the international market
Sugarbeets are cultivated, as a warm and cool season crop, between 30° and 60°N in
Europe, Asia, North America, North Afiica and in small areas in South America.
Following the discovery by Marggraf in 1747 that beets contain sucrose, Achard
developed a process for sucrose extraction and recovery fi'om beets (W'mner, 1993). Mass
selection carried out in fodder beet fiom 1786 to 1830 by Achard and the von Koppy
family improved the sugar content of sugarbeet fiom 6% to 9% (Smith, 1987). Presently,
sugarbeet hybrids can approach 20% sucrose, or 19.1 tons of sucrose per hectare (in
California) (Karma and Lemaux, 1996) under favorable growing conditions. Although
advancements in cultural practices are partially responsible for these improvements, plant
breeding has also played an important role in enhancing sugarbeet productivity.

Traditional methods for breeding sugarbeet are slow. New genetic manipulation
techniques are being developed in order to isolate superior genotypes with favorable
production characteristics and combining ability more rapidly and specifically. The in vitro
techniques of shoot culture cloning, somatic cell selection, somaclonal variation, and
gynogenesis have been developed for more eficient sugarbeet improvement (reviewed by

Saunders et al., 1990). Currently, genetic transformation, and to some extent, somatic cell

2
selection, are the biotechnological vehicles for genetic improvement, especially for “stop

loss” purposes, such as genetic resistance to pests and to herbicides.

Since progress using traditional plant breeding can be relatively slow, tissue culture has
played an increasingly important part for plant breeding. Initially, difl‘erent methods of in
vitro multiplication were used in breeding programs. During subsequent years, progress
was made in developing protocols for sugarbeet shoot regeneration with various systems,
including organogenesis and somatic embryogenesis for use in somatic cell selection,
protoplast manipulations, ovule culture, and genetic transformation Current interest in
sugarbeet somatic embryogenesis is related to proprietary procedures for gene transfer as
well as to prospects for inexpensive field-oriented vegetative propagation systems for seed
production or final crop establishment. The use of protoplasts as a tool in sugarbeet
improvement has been refined in a novel way and applied to gene transfer in what may be
the most eficient transformation system presently available. The quest to achieve reliable
and eficient shoot regeneration that is genotype independent, if not long-term, contimres

to be critical for routine genetic transformation and somatic cell selection

Adventitious shoot regeneration

Sugarbeet is somewhat unusual in that adventitious buds and shoots often form directly
on in vitro petioles of in vitro treated with origin N6-benzyladenine (BA). Early reports
documented direct adventitious shoot regeneration on petioles or midribs of old leaves of
in vitro shoots multiplied on 0.25 mg/L BA supplemented medium (Hussey and Hepher,

1978), from leaves of shoot cultures on media containing cytokinin and auxin (Rogozinska

3
and Goska, 1978), fi'om vegetative apexes and cotyledons of young seedlings on medium

with BA or BA & GA (Atanassov, 1980), from flower buds of greenhouse plants on 1/2
MS (Murashige and Skoog, 1962) medium supplemented with 10 umol/L BA (Miedema,
1982), from shoot cultures from floral stalks on MS medium containing 0.25 mg/L BA
(Saunders, 1982), and fi'om inflorescence tips of field grown sugarbeet and wild beet
(Beta maritima) plants on MS medium with 1 mg/L BA (Zhong et al., 1993). High
ficquencies of direct bud regeneration were observed from petioles of in vitro shoot
cultures on several media containing mixtures of hormones, including 3 mg/L BA, 3 mg/L
1-naphthalene acetic acid (NAA) and 1 mg/L 2,3,5-triiodobenzoic acid (TIBA) for
induction, and 3 mg/L BA and 3 mg/L NAA for regeneration (Detrez, et al., 1988), 0.4
mg/L BA and 0.1 mg/L indole-3-acetic acid (1AA) (Freytag et al., 1988), and 10 mg/L BA
(Ritchie et al., 1989). In red beet (Beta vulgaris), multiple adventitious shoot formation
was observed from single cells of the upper epidermis of petioles of cultured plantlets on
MS medium containing several combinations of cytokinins with other growth regulators
(Harms et al., 1983). The common component in all these media is BA, a strong synthetic
cytokinin.

Adventitious buds arising from explants of other species have been suggested as a
means of clonal propagation (Miedema, 1982; Rogozinska and Goska, 1978).
Nevertheless, Van Harten et al. (1981) found that adventitious buds were a source of
somaclonal variation in some species, for example, in potato. Therefore, somaclonal
variation might also occur in sugarbeet via adventitious buds. Low levels of triploids and

tetraploids in sugarbeet regenerated directly from blade and petiole explants of diploid

4
donor plants were found by Slavova et al. (1982). Abdel-Latif and Sarmders (1985)

detected no somaclonal variation in sugarbeet plants fi'om adventitious buds. Nevertheless,
diploid plants have been regenerated directly fi'om explants of tetraploid plants (Zagorska

and Atanassov, 1985).

Multiplication of axillary shoots
In general, axillary shoots have been multiplied on media with BA Axillary shoots are

rooted on a medium containing an auxin. Margara (1970) first reported that sugarbeet was
grown in vitro and shoots have been induced from the flower buds. Hussey and Hepher
(197 8) indicated that axillary shoots were induced from the axils of the older leaves of in
vitro seedlings on media with 0.12 or 0.25 mg/L BAP. Miedema et al. (1980) reported
that axillary shoots grew out very rapidly when petiole explants fi'om greenhouse grown
plants with axillary buds attached were placed on MS media containing sucrose.
Coumans-Gilles et al. (1981) observed multiplication of axillary shoots from in vitro
culture of inflorescence pieces on a medium containing IBA and BA Miedema (1982) also
reported that shoots were multiplied by axillary bud proliferation on a medium with 1.0
umol/L BA, after the adventitious shoots were initiated from flower buds. In fact, the
technique of multiplication of existing shoot meristems by stimulating wrillary shoot
development was not successfirl with all genotypes (Miedema, 1982), since in some
genotypes, their shoots were stunted or had thick and large leaves. Saunders (1982)
observed the formation of generative shoots during plant regeneration fi'orn axillary buds

on MS medium containing 1.1 uM BA Majewska-Sawka and Jassem (1988) observed

5
that 2 mg/L BA in the medium was better for wild genotypes of the Section Corollinae

and caused fast shoot culture initiation from flower-stalk tips.

Shoot regeneration from callus or suspension cultures

In general, callus requires an exogenous source of auxin and/or cytokinin for induction,
growth and subsequent organ development. Hormone-autonomous cells or callus are
capable of synthesizing part or all of the hormones to support growth, without needing
exogenous auxin and/or cytokinin. De Greef and Jacobs (1979), and Kevers et al. (1981)
obtained nonorganogenic habituated callus lines from leaf pieces which had been initiated
on auxin plus cytokinin media, then subcultured on a basal medium without hormone.
Saunders and Daub (1984) reported that hormone-autonomous callus, which was initiated
in high fi'equency fi'om shoot cultures on MS medium with 0.25 mg/L BA, sustained
growth on hormone-free medium with no reduction in growth rate for at least 6 months,
and it regenerated shoots as well. Callus was initiated fi'om leaf discs of several germplasm
sources on hormone-flee MS medium (Doley and Saunders, 1989), with mean callus
initiation time elevated to 96.7 days, which was considerably longer than was the case
when 1.0 mg/L BA was included in the initial medium (4-6 weeks). Moreover, shoots
were regenerated on the hormone-flee medium without subculture of the callus (Doley
and Saunders, 1989). Masuda et al. (1988) also indicated that hormone-autonomous callus
was initiated fiom young leaf explants in liquid MS medium with 0.25 mg/L BA Then,
following transfer to hormone-flee liquid medium, cells could further divide and grow

autonomously in suspension cultures. Moreover, Van Geyt and Jacobs (1985) reported

6
that habituated (hormone-autonomous) cells were obtained following a gradual reduction

in exogenous auxin and cytokinin levels, but subsequently the cells dedifl‘erentiated and
lost regeneration capacity. Hagege et al. (1991) indicated that habituated sugarbeet callus
and cell suspensions exhibited a shorter linear growth phase, although they divided
actively. The habituated cell lines were defective in cell expansion, cell difi'erentiation and
cell wall development under microscopic observations. Such cultures exhibit all the
characteristics of vitrified tissue with much more watery. The peroxidase activities of
those cell lines were very low (Hagege et al., 1990). Moreover, they produced much less
ethylene than normal (auxin and cytokinin-dependent) callus (Gaspar et al., 1988, Hagege
et al., 1990). Gaspar et al. (1992) also reported that habituated callus lines exhibited many
characteristics of vitrified tissues. The vitrified shoots became habituated under
micropropagation, then progressively lost the organogenic capacities.

Enomoto and Ohyama (1985) reported callus induction and shoot formation fiom in
vitro petioles on various combinations of BA (0.1-5.0 mg/L) and NAA (0 or 0.1 mg/L).
Saunders and Shin (1986), and Saunders and Doley (1986) developed a very simple
system for obtaining callus and subsequent high-frequency shoot regeneration of several
um'elated genotypes, using leaf explants on MS medium containing 1 mg/L BA This high
fiequency but unconventional system was based on hormone-autonomous callus. Further,
Owens and Eberts (1992) modified the original protocol of Saunders and Doley (1986) for
regenerating shoots fiom leaf explant callirs by plating multiple leaf discs (ten 4—mm
discs/plate) on RV medium (an enriched modification of MS medium) containing 1 mg/L
BA and solidified with 0.3% Gelrite, and by transferring the discs to flesh medium every

7
two weeks during the first month. Thereby, the modified protocol produced more calhrs

per plate and higher rates of morphogenesis per unit dry weight of callus than did the one-
step, and one disc/plate methods of Saunders and Doley (1986).

Yu (1989) examined callus induction and difl‘erentiation from leaf explants of 16
germplasm sources of the Genus Beta on MS medium containing BA The leaf explants
from most Beta species exhibited optimum callus formation and shoot difl‘erentiation at
31°C. A large amount of callus was induced in B. vulgar-is L., B. maritima L., B.
intermedia Bung, and B. macrocarpa Guss, but shoots were only regenerated in B.
vulgaris and B. macrocarpa.

Hooker and Nabors (1977) reported that the anti-auxin TIBA in combination with
exogenous BA promoted shoot initiation fi'om cotyledon and hypocotyl callus, but the
regeneration rate was low. However, Tetu et al. (1987) found that high percentages
(52%) of shoot formation occurred with combinations of TIBA and BA Doley (1990)
also reported that TIBA stimulated shoot regeneration fi'om subcultured callus of REL-1.
Abe et al. (1991) indicated that 2 uM TIBA promoted shoot morphogenesis fi‘om callus
culture (from the shoot region of the germinated seed) in the presence of 5 uM BAP. The
combination of TIBA and BAP was also found to be the most favorable for callus
induction fiom hypocotyl explants derived fi'om 21 day-old-seedlings (Jacq et al., 1992),
although the combination gave only a low callus formation fi'om leaf explants flour 6-
month-old plants (Roussy et al., 1996).

Recently, Roussy et al. (1996) reported that the type of cytokinin is important factor for

callus induction in sugarbeet, since callus induction fi'equency was significantly higher on a

8
medium containing 2.2 mg/L thidiazuron (TDZ) medium than on one containing 1 mg/L

BAP.

Callus production for shoot regeneration was afl‘ected by explants and/or genotypes
(Saunders and Doley, 1986; Ritchie et al., 1989; Catlin, 1990). Saunders and Doley
( 1986) mentioned that smaller less expanded leaves were more responsive than larger
leaves fiorn the same plant, and blade explants were superior to petiole explants for callus
induction with the more expanded leaves. Therefore, they proposed that the critical leaf
size may difi‘er with various genotypes and might be associated with the incompleteness of
leaf expansion. Genotype and medium interaction may also afl‘ect in vitro responses. Jarl
and Bomman (1986) observed significant genotype and medium interaction for callus
growth fiom 15 sugarbeet genotypes. Shirnamoto et al. (1993) achieved shoot
regeneration from fiiable calli initiated fi'om the shoot region of the germinated seed for
eight of the 21 sugarbeet germplasms.

Actually, genotype plays a key role in organogenesis and embryogenesis, for calhrs
initiation, shoot regeneration and, to a lesser extent, root formation are afi‘ccted by
genotype. REL-1, an especially useful and tissue-culture fiiendly genotype, was released
to the public by J.W. Saunders in 1987. REL-1 has a heterozygous dominant annualism
nature, which allows it or its direct tissue culture genetic manipulation products to flower
much more rapidly than the ordinary biennial beet the farmer grows. Another clone, REL-
2, was developed specifically for superior embryogenesis and shoot regeneration for

application in biotechnology, REL-1 being one of its parents.

9
Miscellaneous use of callus or suspension cultures

Welandcr (1976) mentioned that callus growth initiated from hypocotyl explants could
be affected by varying nitrogen and sucrose concentration in the media but the influence
could not clearly be related to any particular IAA level. The nitrogen content of the callus
tissue was higher when nitrogen concentration in the media was increased. Nevertheless,
the nitrogen content of the tissue decreased when the sucrose level was risen

Zelmer and Gunther (1988a) examined the activities of glutamate dehydrogenase
(GDH), glutarnine synthetase (GS) and glutamate synthase (GOGAT) from the suspension
cultures of Beta vulgaris and Chenopodr’um album. The level of the enzymes was not
change during the exponential growth of the cell culture. GDH and GS activities reached
a high level, the value of GOGAT was found to be approximately 1112 the amount of GS.
Adding twice the amount of nitrate to the suspension medium, depressed growth,
stimulated GS activity, and reduced GDH activity, adding twice the amount of ammonia,
stopped cell growth, increased GDH activity, and eliminated GS activity. In a follow up
study, Zelmer and Gunther (1988b) firrther reported that the inclusion of 13 herbicides in
the suspension medium caused an inhibition of the enzymatic activities of GDH and a
stimulation of GOGAT within 7 days after herbicide application.

Coutts et al. (1994) detected elicitation of stress-induced proteins in suspension-
cultured sugarbeet cells. Phenylalanine ammonia lyase (PAL) activity increased afier
treated of the suspension-cultured cells with elicitors (including cell wall extracts of
Botortr’s cinerea or chitosan oligomers). The suspension cells responded to these elicitors

by inducing the synthesis of more than 10 polypeptides.

1 o
Eckermann and Baumann (1995) demonstrated enzymatic changes in callus cultures of

sugarbeet during the transition from photoheterotrophic to photoautotrophic growth. A
decrease in the sucrose level of the medium caused an increase in Calvin cycle enzyme

activity, whereas the activity of dissimilation related enzymes either decreased or was not

significantly altered.

Somatic embryogenesis

One of the most dramatic achievements in plant tissue culture has been the discovery of
the phenomenon of somatic embryogenesis. It demonstrated the totipotency of cells of
higher plants. Somatic embryos, derived fi'om somatic cells, are capable of developing
into complete plantlets via a series of developmental stages and morphological changes,
i.e., proembryo, globular, heart- and torpedo-shaped, as well as matru'e embryo. Somatic
embryogenesis is strikingly similar to morphogenesis occurring after the fertilization of the
egg cell. Somatic embryos have been induced from callus, suspension culture cells, zygotic
anbryos, or leaf and stem explants of many species. Multiple embryos form in many cases,
under appropriate culture conditions, with genotypes and growth regulator dependency
being common. The large-scale production of somatic embryos for many high-vahre crops
is now becoming possible. However, only a few reports are available on somatic
embryogenesis in sugarbeet, mostly anecdotal. Atanassov (1976) first mentioned somatic
embryo occurrence fi'om suspension cultures in sugarbeet. Tetu et al. (1987) concluded
that multiple hormonal sequences were necessary for the induction and development of

somatic embryos from calli derived fi'om petiole explants. Freytag et al. (1988) noticed

11
somatic embryogenesis at a low fi'equency from callus (initiated from petioles of cultured

shoots) in all six sugarbeet germplasm sources tested, using a highly supplemented
medium containing 10 vitamins, 6 amino acids, and a combination of indole-3-butyric acid
(BA) and BA Only 2 % of the callus pieces formed somatic embryos, although most
embryos developed into plantlets. Pedersen and Andersen (1988) reported that
embryogenic callus was induced on PGO medium (De Greef and Jacobs, 1979) containing
BAP, and the somatic embryos proliferated on 1/3 MS medium with BAP, IAA, and 6A3.
Whether all these hormones were necessary is unclear. Doley and Saunders (1989)
showed the simple production and partial germination of somatic embryos from leaf disc
callus of a fodder beet cultivar without the use of growth regulators, although embryos
were not seen for at least three months on the initial medium. Kubalakova (1990) obtained
fiiable embryogenic callus at the base of shoot tip explants transferred onto MS or PG.
media without hormones, or on media containing BAP and kinetin The embryogenic
callus maintained its embryogenic ability for three years on hormone-free medium.
Somatic embryos were induced fi'om immature zygotic embryos by Tanning et al. (1992),
who observed some directly secondary embryogenesis from the primary somatic embryos
on MS medium containing 1 mg/L 2,4-D. D’Halluin et al. (1992) used embryogenic callus
(which was initiated from swdlings on PG. medium containing BA, IAA and GA;) in an

Agrobacterium-mediated genetic transformation system for sugarbeet.

Ovule culture

Gynogenesis has been the most efi‘ective technique for obtaining haploids in sugarbeet.

12
Haploid plants were obtained by Hosemans and Bossoutrot (1983) through gynogenesis

by culturing mature excised ovules from male-sterile plants, with a haploid recovery
frequency of only 0.23 %, based on initial number of ovules. In that case, 2.1 % ovules
gave rise to calli or embryos, then 10.9 % of the structures developed into haploid
plantlets after one or two months. Some of the roots on these haploid plantlets were
uniformly diploid. In subsequent work, plants were recovered from 0.17 % of the
unpollinated ovules (Bossoutrot and Hosemans, 1985). In that study, shoot meristems
were haploid, but root tips were polyploid. Van Geyt et al. (1987) reported that up to 2.2
% of cultured ovules from male-fertile and male-sterile plants yielded haploid plantlets.
The fi'equency of haploids was increased to 6.1%, when callus initiated fi'om ovary culture
was transferred to a medium containing 0.5% activated charcoal without 2,4-D. Thus,
ovary culture may lead to haploid sugarbeet plants more eficiently than excised ovule
culture. D’Halluin and Keimer (1986) showed that the variability in haploid recovery rate
was afi‘ected by genotype, and the maximum response frequency achieved was 26%.
Doctrinal et al. (1989) obtained haploid plants by in vitro culture and induction of
embryogenesis of unpollinated ovules. In such gynogenesis, the rate of embryo induction
and haploid plant regeneration was afl‘ected by both genotype and medium Galatowitsch
and Smith (1990) observed that haploid, diploid and tetraploid plants were regarerated
from unfertilized ovule callus of diploid sugarbeet on MS medium. In addition, the
vitrification of shoots was reduced when Gamborg’s BS (Gamborg et al., 1968) salts were
used instead of MS salts in the regeneration medium. Ferrant and Bouharmont (1994)

reported that haploid plants were obtained by gynogenesis directly via embryogenesis fi'om

13
ovules on N6 medium containing IAA and kinetin, or BAP only. They also indicated that

maturity of the female gametophyte was reached a few days prior to anthesis and therefore
ovules fiom unopened flowers were already suitable for plating. A firrther study of the

haploid cells of sugarbeet embryo sac during the first week of in vitro culture showed that

the viable gynogenetic embryo originated only from the egg cell.

Anther culture

Regeneration of haploid plants has an important application in sugarbeet breeding
Various methods have been tried in order to obtain androgenic haploids in sugarbeet.
Banbe and Tanabe (1972) induced only one single, non-rooted diploid shoot. Rogozinska
et al. (1977) observed some regenerated shoots from one callus that was initiated from
anther culture. Unfortunately, all of the regenerants were diploids, quite likely fi'om
somatic tissue, and no regeneration of haploid plants was obtained from anther culture.
Van Geyt et al. (1985) indicated that macrocalli and nuclear division could be induced
fi'om anther cultures and fiee microspore cultures, but no plant regeneration was reported
Tlnrs, so far, the recovery of haploid plants through androgenesis has not proven

successful.

Protoplasts and somatic hybridization
Protoplast hybridization could be considered to study somatic cell genetic manipulations
in sugarbeet. The major disadvantage of somatic hybridization for application in crop

improvement has been the dificulty in achieving plant regeneration from protoplasts of

14
many plant species. In some early studies, no colony was formed fiom protoplasts which

were isolated from leaves of greenhouse-grown sugarbeet plants (Nam et al., 1976;
Binding et al., 1981; Bomman et al., 1982). Subsequently, callus colonies were induced
from protoplasts of a sugarbeet cell suspension culture, but no plant was regenerated fiom
protoplast derived cultures (Szabados and Gaggero,l985; Bhat et al., 1985). Bhat et al.
(1986) only succeeded in regenerating roots (no shoots) from protoplast calli derived fi'om
shoot culture leaves in three diverse beet genotypes. None of these reports mentioned use
of genotypes with demonstrated shoot or embryo regeneration capacity. Steen et al.
(1986) only mentioned that the variability was found in regenerated plants fi’om the
protoplasts of the tetraploid line 68-3, but no detailed procedure for regenerating plants
from protoplasts was provided. Krens et al. (1990) found that the inclusion of n—propyl
gallate (a lipoxygenase inhibitor) in the isolation and culture media was audal for cell
division, colony formation and regeneration, although mesophyll protoplasts of sugarbeet
showed a significant decrease in protoplast viability just one day after isolation.
Nevertheless, plating eficiencies were still very low (fi'om 0.005% to 1%). Subsequently,
embedding protoplasts in alginate was found to increase the plating eficiency
(Schlangstedt et al., 1992; Hall et al., 1993; Pedersen et al., 1993). In fact, the plating
eficiencies of protoplasts fi'om leaf mesophyll tissue was low and showed substantial
variance (overview by Schlangstedt et al., 1994). Ritchie et al. (1989) proposed that sub-
epidermal petiole parenchyma protoplasts might preferentially permit shoot regeneration
Schlangstedt et al. (1994) demonstrated that much higher plating eficiencies were

obtained fi'om petiole protoplasts (average 4.6%) than from mesophyll protoplasts

15
(average 1%). In addition, a higher pr0portion of protoplasts with doubled DNA content

(i.e., nuclei in the GZ/M stage of the cell cycle) was found in petiole protoplast
populations via DNA histograms. However, no plantlets were obtained. Later, Lenzner et
al. (1995) succeeded in regenerating plants from mesophyll protoplasts isolated fiom 2- to
3-week-old subcultured shoot cultures (instead of the methods described by Krens et al.
(1990) using mesophyll protoplasts fi'om seedlings). The mesophyll protoplasts fi'om in
vitro shoots showed high plating emciencies up to 4%, then developed into callus. Two of
the lines with fiiable callus containing starch grain cells fi'equently formed adventitious
shoots in the light after cytokinin (BAP or TDZ) treatment (Lenzner et al., 1995).
Recently, a high frequency of regeneration of plants was obtained via protoplast culture,
and the technique was further applied to gene transformation (Hall et al., 1996). The
protoplasts were isolated specifically from stomatal guard cells. The cells are the only
progenitors of totipotent protoplasts in sugarbeet leaves, it was claimed, and were
separated by using computer-assisted microscopy (Hall et al., 1995). Overall, many
factors may influence shoot regeneration fiom protoplasts, such as genotype, source of
explants for isolating protoplasts, isolating procedures, cultural conditions, media, plating

density, and environment.

Somaclonal variation
Somaclonal variation in sugarbeet has been reviewed by Saunders et al. (1990).
Somaclonal variation includes genetic or epigenetic change via in vitro procedures.

Somaclonal variation arises during the tissue culture phase or fiom preexisting genetic

16
variation within the explants. Reducing the length of the cell culture phase could reduce

the occurrence of somaclonal variation. In sugarbeet, polyploidy and aneuploidy have been
detected soon after the initiation of callus (Atanassov et al., 1978). Saunders and Doley
(1986) found prolific regeneration fiom leaf disc callus on MS medium with 1 mg/L BA in
a somaclonal variant. Steen et al. (1986) reported that within genotypes the frequency of
variation was higher in plants regenerated fiom protoplasts than in plants regenerated fi'om
suspension cultures. In addition, the degree of variation was higher from suspension
cultures than from callus. Yu (1987) observed that some regenerant plants fiom leaf disc
callus displayed difi‘erences in leaf morphological and pigmentation. Plants of 36 and 38
chromosome number were obtained from 18 and 19 chromosome plants, respectively.
Mixoploid plants containing roots with either doubled or undoubled chromosome number
were also detected. Yu et al. (1991) further reported that polyploid regenerants usually
developed wider leaf blades, while diploid regenerants developed smaller leaf length to
width ratios. In one experiment, 73.5% of the experimental plants contained the 19-
chromosome number of the donors, 23.1% of the plants had roots with doubled

chromosomes, and 2.6% had mixoploids with sectors of diploid and tetraploid roots in the

same plant (Yu et al., 1991).

Somatic cell selection
Somatic cell selection has contributed to crop improvement in sugarbeet. Somatic cell
selection involves the application of some chemical or physical agents or environment to

cultured cells or tissues with the intention of favoring the survival or multiplication of

17
certain variant cells in the cell population. For instance, stable resistance could be obtained

by applying a herbicide concentration lethal to normal cells. Saunders et al., (1992)
successfully recovered monogenic dominant sulfonylurea-resistant sugarbeet plants fiom
somatic cell selection. They reported that acute somatic cell selection for sulfonylurea
herbicide resistance involved unmutagenized cell clusters of clone REL-l plated on MS
agar medium with 2.8 nM chlorsulfuron and 1 mg/L BA Shoots regenerated fi'om the
only surviving colony without subculture. Those shoots were further resistant to 28 nM
chlorsulfirron incorporated in the rooting medium, a concentration that killed similar
shoots of REL-1. Shoot resistance to chlorsulfirron in this single gene mutant, designated
CRl-B, was 300 to 1000- fold greater than in REL-l. In greenhouse studies, CRl-B was
cross-resistant to some other sulfonylurea herbicides tested (Hart et al., 1993). Two
imidazolinone herbicide-resistant sugarbeet mutants, 93R30B and Sir-l3, have been
independently generated via somatic cell selection against the imidazolinone herbicide
Pursuit (according to the system of Saunders et al., 1992) and prevent imidazolinone
carryover injury to sugarbeets (Wright, 1997). Irnportantly, the mutants recovered fiom
somatic cell selection do further express the resistance in whole plants, as well as transmit
the resistance sexually for use in development of resistant hybrid cultivars of sugarbeet.
The 93R30B mutant was recovered in a genotype bred for somatic cell selection as well as
homozygous dominant for the earlier sulfonylurea resistance allele, and proved to be a
second somaclonal mutation on the same allele (Wright, 1997). The imidazolinone-

resistant mutants are currently being incorporated into commercial breeding lines.

1 a
Genetic transformation

Genetic engineering techniques have been used to overcome some factors limiting
traditional plant breeding including barriers among species. Nevertheless, sugarbeet has
been considered one of the most recalcitrant plant species to molecular methodologies.
Part of the apparent recalcitrance has been due to the proprietary nature of much of the
sugarbeet transformation research. Fortunately, these limitations involving low
transformation rates and poor regeneration efliciencies appear to have been overcome by
Hall et al. (1996), who have established a striking breakthrough system in the molecular
manipulation of sugarbeet.

Lindsey and Jones (1987, 1989) first reported that gene expression and stable
transformation could be obtained by electroporation-mediated protoplast transformation in
sugarbeet, but the regeneration of transgenic plants via this system was limited due to
technical dificulties in regeneration from protoplasts.

Wang et al. (1985) and Paul et al. (1987) demonstrated that sugarbeet is susceptible to
infection by bacteria, such as Agrobacten’um tumefaciens wild type nopaline strains and A.
rhizogenes. Harpster et al. (1988) produced stably transformed callus by using A.
tranefirciens, but no regeneration of plants was obtained. Krens et al. (1988) recognized
that optimal transformation fiequencies were dependent on sugarbeet genotype and
Agrobacterium strain. They found that using Agrobacterium shooter mutants, or strains
carrying an isolated cytokinin gene, in order to afl‘ect the endogenous hormone ratios
prevented shoot regeneration fiom the transformed calli. Successfirl regeneration of

transformed sugarbeet plants by using A. trancfaciens was reported by Lindsey and Gallois

19
(1990). Binary vectors were used to carry selectable (NPTII) and screenable genes (CAT

and GUS). The use of cultured shoot-base tissues allowed rapid and prolific generation of
putatively transgenic plants while avoiding a callus phase. This transformation rate was
dependent on the explant sources, genotypes, and selection conditions. D’Halluin et al.
(1992) developed Agrobacterr‘um-mediated transformation from seedling embryogenic
callus to recover transgenic plants with herbicide resistance by introducing either the
bialaphos resistance gene (bar) or a gene encoding mutant acetolactate synthases (ALS).
The transformants expressing the ALS gene were resistant to field levels of sulfonylurea
compounds applied in the greenhouse, whereas the progeny of transformants expressing
the bar gene were resistant to commercial levels of glufosinate-ammonium applied in the
field.

In order to increase the level of gene expression in sugarbeet, Ingersoll et al. (1996)
demonstrated that various types of promoters (osmotin, PR-S, pinz, and CM 358) that
were fused to the gas coding region and biolistically transferred into sugarbeet suspension
cells did affect the level of expression The expression of osmotin promoter-leader
construct was the highest, about 2.5-fold higher than for 35S-gus. Expression of PR-S-gus
and pinZ-gus were intermediate but somewhat higher than 35S-gus.

Hall et al. (1996) achieved high transformation fiequencies by using a polyethylene
glycol (PEG)-mediated protoplast transformation technique. The protoplasts were derived
fi'om stomatal guard cells, which Hall et al. (1996) assert are the only progenitors of
totipotent protoplasts in sugarbeet leaves. Transgenic plants resistant to glufosinate-

ammonium-based herbicides were obtained within only 8 to 9 weeks. This system seems

2 o
to ofl‘er highly eflicient regeneration and transformation as well as rapid development,

although the genotype independence claimed was not rigorously tested. This optimized
protocol can be applied to integrate useful genes in order to attain eflicient transformation
for genetic modification with herbicide resistance, disease resistance, and enhanced yield

traits of sugarbeet in the near firture.

So far, researchers have established good systems for superior somatic embryogenesis
and shoot regeneration abilities in a few sugarbeet genotypes. These systems should be
further developed to attain (1) eficient gene transformation for genetic modification and
improvement, (2) mass propagation of male-sterile lines or of elite F1 plants with field
delivery capability for use as artificial seeds, and (3) novel schemes for somatic cell

selection.

The general objectives of this research are to:
(1) determine the level of embryo production from suspensions of two genotypes, REL-1
and REL-2, plated onto MS media with combinations of ABA with difi'erent
concentrations of grth regulators, sucrose, or difl‘erent sole nitrogen sources, (2) detect
the germination and conversion rates of difl‘erent length categories of somatic embryos,
(3) examine the germination and conversion rates of somatic embryos encapsulated with
alginate in MS medium or in H20 afier difi‘erent periods of cold storage at 4 °C, (4)
determine if nitrate, ammonium, glutamine, glutamate, urea, proline, glycine betaine, and

choline can be utilized as sole nitrogen sources for growth of suspension plate-out, shoot

2 1
culture, and induction of leaf disc callus and subsequent shoots of sugarbeet clone REL-1,

(5) test glutamine, glutamate and glycine betaine for use as either sole carbon or carbon
& nitrogen source, in order to ascertain their potential role in somatic cell selection
schemes, and (6) examine the specificity of inhibition by nitrate uptake inhibitor

phenylglyoxal on callus growth with either nitrate or ammonium alone and in combination

22

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.. ...... .32... .
. ... ...arrr

 

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30
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CHAPTERI

SOMATIC EMBRYOS FROM CALLUS OF SUGARBEET
BIOTECHNOLOGY CLONE REL-l

ABSTRACT
Somatic embryos could be used for proliferative propagation or for gene transfer

procedures in sugarbeets (Beta vulgaris L.) if adequate methods for initiation and development

could be devised. With sugarbeet model clone REL-1, plating of flesh suspension culture cells
grown with hormone-free Murashige and Skoog (MS) medium onto further hormone-flee MS
medium elicited a low frequency of somatic embryogenesis, about one embryo per ml of
suspension used. The inclusion of 0.1 or 0.3 mg/L abscisic acid (ABA) in the plate-out medium
increased the number of somatic embryos in this system A combination of naphthaleneacetic
acid (NAA) (1 mg/L) and ABA (0.1 mg/L) gave the highest somatic embryo yield, 15 embryos
per ml of suspension. After 22 to 40 days, embryos at various stages, ranging fiom globular,
heart, and torpedo-shaped embryos to mature opaque white embryos with cotyledons and
radicles, were present at the callus surface. The external morphology of several somatic
embryos was examined by scanning electron microscopy. The somatic embryos developed into
normal plantlets, exhibiting betalain pigmentation on hypocotyls after being transferred onto
horrnone-free MS medium. The conversion rate of somatic embryos of difl'erent lengths (1, 2, 3

mm) into complete plantlets was similar (78, 81, and 85 % respectively). The conversion rate

31

32
of encapsulated somatic embryos was 75 % with 1.5 % alginate in MS medium and 72 % with

1.5% alginate in H20.

INTRODUCTION

Somatic embryogenesis is the process of embryo initiation and development from somatic,
i.e., non-germ, cells. In plant tissue culture, somatic embryos have most often been produced
from callus or suspension culture cells under appropriate conditions, with genotype
dependency being common. Somatic embryos are under investigation in species such as alfalfa
and celery for use in production of artificial seeds (Gray and Purohit, 1991). Elite highly
productive individual genotypes from genetically heterogeneous cultivars, or superior-
combining male sterile clones, can be vegetatively reproduced on a large scale as somatic
embryos and delivered to the field in a seedlike fashion following conditioning and coating, or
alternatively, by fluid drilling. Somatic embryos also could be used in genetic transformation
applications as exemplified in gene transfer with Agrobacterium in walmrt (McGranahan ct
al.,l990), gape (Le Gall et al., 1994) and Datura tnnoxia (Ducrocq et al., 1994). Synthetic
seed production, as well as gene transfer, would be more eficient if somatic embryogenesis
also occurred via immediate subsequent cycles wherein multiple new embryos arose fiom
existing ones. This self-replicating production is termed secondary somatic embryogenesis.

Somatic embryos in sugarbeet were first reported by Atanassov (1976) in suspension
cultures. Tetu et al. (1987) concluded that multiple hormonal sequences were necessary for the
induction and development of somatic embryos from callus. Somatic embryogenesis at a low

fiequency in callus was reported by Freytag et al. (1988) in all six germplasm sources tested.

33
Kubalakova (1990) described callus from at least one genotype that had maintained its

embryogenic ability for three years on media without gowth regulators. Somatic embryos
were directly produced on sugarbeet zygotic embryos by Tenning et al. (1992), who also
observed some direct secondary embryogenesis. Doley and Saunders (1989) reported the

simple production and partial germination of somatic embryos fi'om leaf disc callus of a fodder
beet cultivar without the use of gowth regulators. D'Halluin et al. (1992) used embryogenic
callus fi'om seedlings in an Agrobacterium-mediated genetic transformation system for
sugarbeet.

REL-1 is a self-fertile diploid sugarbeet clone bred for ease of tissue culture manipulations.
It exhibits a high fiequency of leaf disc callusing, shoot regeneration fi'om hormone
autonomous callus, dispersed suspension cultures and resistance to shoot vitrification REL-1
is also heterozygous for monogermness (Mm), annualism (Bb), and red hypocotyl (Rr).
REL-1 has been used for the recovery of monogenic dominant sulfonylurea herbicide resistance
by somatic cell selection (Saunders et al., 1992) and of other mutant traits (Saunders et al.,
1990).

We report here (1) the initial mccess in obtaining somatic embryos fi'orn biotech clone
REL-1, (2) converting somatic embryos into plantlets, (3) examination of later stages of
somatic embryos under scanning electron microscopy, and (4) encapsulation of somatic

embryos in initial efl‘orts to produce artificial seeds.

34

MATERIALS AND METHODS

Plgt materials

All experiments were performed with the diploid sugarbeet (Beta vulgar-11s: L.) clone REL-
], released to the public in 1987 . REL-1 has been maintained in shoot culture (Saunders, 1982)
and is available upon request, either as in vitro shoots, whole plants, or S1 seed.
Culture media

The culture media contained MS mineral salts (Murashige and Skoog, 1962), 100 mg/L
myo-inositol, 1.0 mg/L thiamine‘HCl, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine‘HCl, and 30
g/L sucrose. Media used in plating out were gelled with 3.5 g/L phytagel. The gowth
regulators used were : 6-benzyladenine (BA) (1.0 mg/L), 2,3,5-triiodobenzoic acid (TIBA)
(1.0 mg/L), l-naphthaleneacetic acid (NAA) (0.25-1.0 mg/L), and/or (~_t)cis,trans-abscisic acid
(ABA) (0.1-1.0 mg/L). The pH was adjusted to 5.95 prior to autoclaving. ABA was filter-
sterilized and added into previously autoclaved and partially cooled media Culture vessels
were 125 mL Erlenmeyer flasks or 20 x 100 mm Falcon disposable plastic Petri plates. Medium

volume per vessel was 35 mL. Flasks were closed with foam caps and aluminum foil. Petri

plates were sealed with one layer of Parafilm.
Initiation of gllg m maintenance of: cell sumsions

Callus was initiated from leaf discs (8 mm diameter) fiom partially expanded leaves of
geenhouse gown REL-1 on MS medium with 1 mg/L BA (Saunders et al., 1992) and 0.9 %
DifcoBactoagarinPetri platesat30Cinthedark, onediscperplateof35mLmedium.
Callus was first seen after one month, and after another month, 2 to 3 g ofleaf-disc callus was

transferred to hormone fiee liquid MS medium in 125 mL Erlenmeyer flasks for gowth at

35
21:2 °C in the dark. The suspension cultures were shaken on rotary shakers at 125 rpm to

aerate the cultures and to reduce cell cluster size.
Induction of somatic embgzos

After 5-7 days, suspension cultures were subcultured with hormone-flee MS medium for
another 5-7 days (poured the flesh medium into the old suspension flask and mixed, then
divided into two flasks). Suspensions used as inoculum for platings were pushed through a
stainless steel sieve with 830 um openings. Sieved suspension cells were washed with hormone
fiee liquid medium and plated on MS media with no gowth regulators or with combinations of
BA and NAA, or NAA, ABA, and TIBA Each Petri plate received 1 mL of suspension
preparation containing about 0.1g fi'esh weight, and was incubated in dim light (less than 5
umol m"2 s") from fluorescent lamps (Philips cool white econowatt) at 25°C. Minimum size for
a somatic embryo to be counted was 0.5 mm.
Germination and conversion of somatic embryos

Somatic embryos were transferred onto hormone-fies MS medium under light (20-50
umol m’zs‘l from fluorescent lamps) at 25°C. The proportion that developed into normal
plantlets with roots constituted the conversion rate of somatic embryos into complete plantlets.
Scanning elgron migrQscopy

According to the procedures of Flegler et al. (1993) somatic embryos were fixed in 2 %
glutaraldehyde in 0.1 M cacodylate buffer for 1 hour, then washed with two changes of bufl‘er
for 10 minutes each. Dehydration was accomplished by immersion for 10 minutes each in 25,
50, 75, 95 and 100 % ethanol with two additional changes of 100 % ethanol at the end. Fixed
somatic embryos were dried in a Balzers critical point dryer, mounted and sputter-coated with

gold, and examined with a J SM-6400 scanning electron microscope.

36

Encapsulation of somatic embryos
Somatic embryos (approximately 2-3 mm) were encapsulated with hormone-free MS

medium including 1.5 % sodium alginate, or H20 including 1.5 % sodium alginate. A pipette
was used to suck up single embryos with the alginate, which were then dropped into 50 mM
CaClz'Hzo and stirred for 20 minutes, thus formed sodium alginate beads of a size large
enough to cover the entire somatic embryo. Then, the germination and conversion rates of

embryos in each encapsulation solution were determined, after the beads were placed on

horrnone-free MS agar plates.
Datamabm's

Each experiment with 10 replications was performed 3 times with similar results. Analysis
of variance was based on a randomized complete block design. The average mrmber of somatic
embryos per plate for each medium was subjected to ANOVA, and the least significant

difi’erence (LSD) test (P=0.05) was performed to permit individual treatment comparisons.

RESULTS AND DISCUSSION

Inggg'gn 9f gamatig embryos

A low fiequency of somatic embryogenesis, almost one embryo per Petri plate, was
obtained following plating-out of suspension culture cells on hormone-flee MS medium NAA
and BA tested either individually or in combination did not induce production of significantly
more somatic embryos than did the hormone-flee medium. Rather, the presence of BA at 0.5
or 1.0 mg/l markedly reduced the occurrence of somatic embryos (Table 1-1). On the other
hand, ABA at 0.1 or 0.3 mg/l in the media increased the number ofsomatic embryos per plate

up to eight-fold (Figure 1-1). The highest somatic embryo yield of 15 per plate was attained

37
with the combination 1 mg/l NAA and 0.1 mg/l ABA (Figure 1-1). ABA is best known for its

positive efl‘ects in somatic embryogenesis where it normalizes development and inhibits
precocious germination (Ammirato, 1974; 1983; eren et al., 1990). ABA also play a key part
on conversion in somatic embryo systems of alfalfa and carrot (Fujii et al., 1990; Nickle and
Yeung, 1993). TIBA did not stimulate somatic embryogenesis in REL-1 using the described
procedure, even though Doley (1990) found that TIBA promoted bud formation in calhrs of
REL-l. Tetu et al. (1987) reported a stimulatory efi’ect of TIBA on bud formation in sugarbeet
callus of another genetic backgound.
mew—mum

During the first two weeks following plating-out, white or light yellow callus proliferated.
From the third to the sixth week, various late stages of somatic embryos from torpedo to
mature opaque white embryos with cotyledons were clearly distinguishable at the callus surface
(Figure 1-2 and 1-3). The simultaneous occurrence of embryos of difi‘erent lengths (0.5 to 4
mm) indicated that somatic embryogenesis was not uniform and synchronous. Afier 40 days,
most of the somatic embryos with cotyledons were around 2-3 mm long. Each somatic embryo
(Figure 1-4) could be easily separated fi'om the surrounding callus.
anvegjon of gmatic @bggs into plantlets

After being transferred onto hormone-free MS medium, the cotyledons and radicles of the
bipolar embryos gadually developed into shoots and roots simultaneously to form normal
plantlets (Figure 1-5). Plantlets usually displayed betalain pigmentation on the hypocotyls, the
colors including yellow, light red or deep red (Figure 1-6). All somatic embryos longer than 2
mm germinated. However, the proportion of conversion into complete plantlets was somewhat

less because of subsequent callusing of somatic embryos or the formation of abnormal plants.

38
The conversion rate of somatic embryos of difl‘erent sizes into complete plantlets was 85 %

(Table 1-2).

Embryo morphology could have afi‘ected subsequent germination and conversion of
somatic embryos into plantlets. Abnormal somatic embryos are common in other species, for
example, caraway (Ammirato, 1974) and soybeans (Buchheim et al, 1989). Achieving a high
degee of normal morphology usually involves optimizing the culture medium components and
environmental conditions.

Scanning electron micrggpy

The development of somatic embryos fi'om REL-1 callus resulted in torpedo stage (Figure
1-7 and 1-8) and mature embryo stage with cotyledons (Figure 1-9) and radicles (Figure l-10).
Although somatic embryogeny is reported to mimic zygotic embryogeny in many respects
(Crouch, 1982; Sharp et al, 1980), this has yet to be demonstrated completely in sugarbeet.
Artschwager (1927) and Artschwager and Starrett (1933) have described the anatomy of
embryo and seed development in sugarbeet. A more detailed comparison of somatic with
zygotic embryogenesis would require detection of earlier stages of somatic embryogeny in
callus or liquid cultures. Fowke et al. (1994) reported that the general morphology of the
mature embryos and zygotic embryos was very similar at the same stage of development in
white spruce, except for some difi‘erences in embryo size, orientation of cotyledons and surface
wrinkling.

Some abnormal somatic embryos with possible vascular connection to the callus (Figure 1-
11), or with swollen hypocotyls (Figure 1-12 and 1-13) were observed on the plate-out
medium. These abnormal morphologies might reduce subsequent conversion of somatic

embryos into normal plantlets.

39
Trichomes could be seen on the cotyledonary surface of some somatic embryos with SEM

(Figure 1-14, 1-15 and 1-16). Goldberg et al. (1994) mentioned that trichomes were not
present on wild-type cotyledons of Arabidopsis seedlings. Nevertheless, trichomes, which were
markers for postembryonic development, occurred on leaf, stem and sepal surfaces in wild-type
Arabidopsis plants (Hulskamp, et al., 1994). The mature leaf trichomes were regularly
distributed in the epidermal layer. On the other hand, Gray et al. (1993) reported trichome
development on precociously germinating cotyledonary stage embryos fiom Cucumis melo.
Encapsulation of somatic embgzos

In some crops, such as alfalfa, celery, and carrot, somatic embryos have been used in efl‘orts
to make artificial seeds (Fujii et al, 1993; Kitto and Janick, 1985). Isolated somatic embryos
(Figure 1-4) of REL-1 were utilized for encapsulation to study the possibility of making
artificial seeds in sugarbeet (Figure 1-17). The somatic embryos encapsulated with 1.5 %
alginate in MS medium resulted in 100 % germination and 75 % conversion; those
encapsulated with 1.5 % alginate in H20 resulted in 100 % germination and 72 % conversion,
in each case after being transferred on agar plates for 20 days (Table 1-3).

This experiment indicated that encapsulation of somatic embryos reduced the conversion
rate (about 10 %) as compared to nonencapsulated somatic embryos (according to Table 1-2
and Table 1-3). The same result was observed by Rao and Singh (1991), who reported that the
conversion rate for encapsulated embryos was significantly lower (49.7 %) as compared to that

of nonencapsulated somatic embryos of hybrid Solanum melongena L..

Based on the research reported here, the model biotech clone REL-1 is capable of at least

moderate intensities of production of somatic embryos, which then are easily collected and

‘40
converted into plantlets. This is the first report of sugarbeet somatic embryos that describes

the use and promotive efl‘ect of ABA on embryo number. In addition, its the first report of the
encapsulation of somatic embryos in sugarbeet. In order for REL-l somatic embryos to be
usefirl in gene transfer research, conditions for secondary somatic embryo production must be
developed. If somatic embryos are to find a direct application in the field, conditions for their
massive proliferation in other genotypes must be found. It is encouraging to note that both

Tenning et al. (1992) and Kubalakova (1990) reported some secondary embryogenesis in

sugarbeet cultures.

REFERENCES

Ammirato, P.V. 1974. The effects of abscisic acid on the development of somatic
embryos fi'om cells of caraway (Carum carvi L). Bot. Gaz 135: 328-337.

Ammirato, P.V. 1983. The regulation of somatic embryo development in plant
cell cultures: Suspension culture techniques and hormone requirements.
Bio/Technology 1: 68-74.

Artschwager, E. 1927. Development of flowers and seed in the sugar beet.
J. Agri. Res. 34: 1.25.

Artschwager, E. and RC. Starrett. 1933. The time factor in fertilization and
embryo development in the sugar beet. J. Agi. Res. 47: 823-843.

Atanassov, A1. 1976. Analysis of organogenetic abilities of contimrously
cultivated somatic tissues of sugar beet on the basis of obtaining single-
celled clones and suspension cultures. In F.J. Novak (ed). Use of tissue
cultures in plant breeding. Institute of Experimental Botany, Olomouc.
pp. 69-80.

Buchheim, J.A, S.M Colburn, and JP. Ranch. 1989. Maturation of soybean
somatic embryos and the transition to plantlet gowth. Plant Physiol. 89:768-
775.

41

Crouch, ML. 1982. Non-zygotic embryos of Brassica napus L. contain embryo
-specific storage proteins. Planta 156: 520-524.

D'Halluin, K, M. Bossut, E. Bonne, B. Mazur, J. Leemans, and J. Botterman
1992. Transformation of sugarbeet (Beta vulgaris L.) and evaluation of
herbicide resistance in transgenic plants. Bio/1‘ echnology 10: 309-314.

Doley, W.P. and J .W. Saunders. 1989. Horrnone-free medium will support callus
production and subsequent shoot regeneration fiom whole plant leaf explants in
some sugarbeet (Beta vulgaris L.) populations. Plant Cell Rep. 8: 222-225.

Doley, W.P. 1990. Genotype and gowth regulator efl‘ects on shoot regeneration
from primary and serially-subcultured hormone-autonomous callus of sugarbeet
(Beta vulgaris L.). Ph.D Dissertation, Michigan State University.

Ducrocq C., RS. Sangwan, and BS. Sangwan-Norreel. 1994. Production of
Agrobacterium-mediated transgenic fertile plants by direct somatic embryo-
genesis from immature zygotic embryos of Datura innoxia. Plant. Mol. Biol.
25: 995-1009.

Flegler, S.L., J.W. Heckrnan Jr., and KL. Klomparens. 1993. Scanning and
transmission electron microscopy. W.H. Freeman, New Yorlg pp. 151-167.

Fowke, L.C., S.M. Attree, and P.J. Rennie. 1994. Scanning electron microscopy

of hydrated and desiccated mature somatic embryos and zygotic embryos of
white spruce (Picea glauca Voss.) Plant Cell Rep. 13: 612-618.

Freytag, AH., S.C. Anand, AP. Rao-Arelli, and L.D. Owens. 1988. An improved
medium for adventitious shoot formation and callus induction in Beta vulgaris
L. in vitro. Plant Cell Rep. 7: 30-34.

Fujii, J.AA, D. Slade, and R Olsen 1990. Alfalfa somatic embryo maturation
and conversion to plants. Plant Sci. 72: 93-100.

Fujii, J .AA, D. Slade, and K. Redenbaugh. 1993. Planting artificial seeds and
somatic embryos. In K Redenbaugh(ed.), Synseeds. CRC Press. Davis,
California. pp. 183-202.

Goldberg, RB., G. Paiva, and R Yadegari. 1994. Plant embryogenesis: zygote
to seed. Science 266: 605-614.

Gray, DJ. and A Purohit. 1991. Somatic embryogenesis in development of
synthetic seed technology. Crit. Rev. Plant Sci. 10: 33-61.

Gray, D.J., D.W. McColley, and ME. Compton. 1993. I-Iigh-fi'equency somatic

42

embryogenesis fi'om quiescent seed cotyledons of Cucumis melo cultivars.
J. Amer. Soc. Hort. Sci. 118: 425-432.

Hulskamp, M., S. Misera, and G. Jurgens. 1994. Genetic dissection of trichome
cell development in Arabidopsis. Cell 76: 555-566.

Kitto, S. and J. Janick. 1985. Production of synthetic seeds by encapsulating
asexual embryos of carrot. J. Am. Soc. Hort. Sci. 110: 227-232.

Kubalakova, M. 1990. Somatic embryogenesis and cytoplasmic sterility in Beta
vulgaris L. var. sacchanfera. Biol. Plant. 32: 414-419.

Le Gall, O., L. Torregosa, Y. Danglot, T. Candresse, and A Bouquet. 1994.
Agrobacterium-mediated genetic transformation of gapevine somatic embryos
and regeneration of transgenic plants expressing the coat protein of gapevine
chrome mosaic nepovirus (GCMV). Plant Sci. 102: 161-170.

McGranahan, G.H., C.A Leslie, S.L. Uratsu, and AM. Dandekar. 1990.
Improved eficiency of the walnut somatic embryo gene transfer system
Plant Cell Rep. 8: 512-516.

Murashige, T. and F. Skoog. 1962. A revised medium for rapid gowth and
bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497.

Nickle, TC. and EC. Yeung. 1994. Further evidence of a role for abscisic acid in
conversion of somatic embryos of Daucus carota. In Vitro Cell. Dev. Biol.
30: 96-103.

Rao, P.V.L. and B. Singh. 1991. Plantlet regeneration from encapsulated somatic
embryos of hybrid Solanum melongena L.. Plant Cell Rep. 10: 7-11.

Saunders, J .W. 1982. A flexible in vitro shoot culture propagation system for
sugarbeet that includes rapid floral induction of ramets. Crop Sci. 22: 1102
1105.

Saunders, J.W., W.P. Doley, J.C. Theurer, and MH Yu. 1990. Somaclonal
variation in sugarbeet. In Y.P.S. Bajaj (ed): Biotechnology in agriculture
and Forestry. Vol 11. Somaclonal variation in crop improvement. I. Springer-
Verlag, Berlin Heidelberg. pp. 465-490.

Sarmders, J.W., G. Acquaah, K.A Renner, and W.P. Doley. 1992. Monogenic
dominant sulfonylurea resistance in sugarbeet fi'om somatic cell selection
Crop Sci. 32: 1357-1360.

43

Sharp, WR, MR. Sondahl, L.S. Caldas, and SB. Marafi‘a 1980. The physi-
ology of in vitro asexual embryogenesis. In J. Janick, (ed). Horticultural
Reviews, vol 1, AVI , Westport, CT. pp. 268-310.

Tenning, P., E.W. Weich, U.B. Kjarsgaard, M.A Lelu, and M. Nihlgard. 1992.
Somatic embryogenesis fi'om zygotic embryos of sugar beet (Beta vulgaris L.)
Plant Sci. 81: 103-109.

Tetu, T., RS. Sangwan, and BS. Sangwan-Norreel. 1987. Hormonal control of
organogenesis and somatic embryogenesis in Beta vulgan‘s callus. J. Exp. Bot.

38: 506-517.

Wilen, RW., RM Mandel, and RP. Pharis. 1990. Efl‘ect of abscisic acid and high
osmoticum on storage protein gene expression in microspore embryos of Brassica
napus. Plant Physiol. 94: 875-881.

44

Table 1-1: The efi'ect of BA and NAA concentrations on somatic embryogenesis.

One mL of suspension cells was plated onto media with various
combinations of BA and NAA and scored 68 days after plating.
Means marked with same letter are not significantly difl‘erent

 

 

 

according to LSD with P<0.05.
Number 91' gmgtic embng [Er 2m

BA BA BA BA

mg/L 0 0.25 0.5 1.0

NAA 0 0.75 ab 0.25 bc 0 c 0 c

NAA 0.25 1.0 a 0.25 bc 0.08 c O c

NAA 0.5 0.67 ab 0 c 0 c 0 c
NAA 1.0 1.08 a 0.08 c 0 c 0.08 c

 

 

 

45

Table 1-2 : Efi‘ect of embryo length on the proportions of somatic embryos
germinating and converting into complete plantlets.

 

 

 

Length of somatic embryos Germination rate Conversion rate
mm % %
(0.5 - 1.9) 88 (61/69) 78 (54/69)
(2.0 - 2.9) 100 (96/96) 81 (78/96)
(3.0 - 3.9) 100 (28/28) 86 (24/28)

 

 

 

Table 1-3: Comparison of encapsulated somatic embryos with MS medium and H20, and
efl‘ects on the germination and conversion rate on MS medium (genotype REL-1).

 

 

 

EneapsulatedwithMS medium& 1.5%alginate EncaparlatedwithI-Igo& 1.5%alginate
Germination rate 100 % (16116) 100 % (18l18)
Conversion rate 75 % (12116) 72 96 (IS/18)

 

 

 

 

46

.86 V m 5? and 3 wEEooon
“:2er 388953 «on one 5:2 08% 5? e815: 2.32 .So

2% sea as 2 <5. as {<2 .<m< no 288558 a? see

8:0 So @033 nonconmsm no 1: non mobs—=0 032:8 no .385: BE. ..2 oSME

 

T <m<n no <3 a ..o <m<l o <93—
:BE. <2» u..- <<z <m< .o 22.3358

 

 

 

o (m; 0 (<2

 

— (m; — ((2 F (mt. and ((2 o (arr F <<z o (0:. mad (<2

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

o

slap cc sans sobqun enemas JO raqumu

N—

v—

2

Figure 1-2: Opaque white somatic embryos (1.5 mm long) on the surface of callus

tissue 22 days after suspension plating.

Figure 1-3: A 3 mm long tricotyledonary somatic embryo 30 days after suspension
plateout.

 

 

Figure 1-4: Isolated somatic embryos (length: 0.5 - 4 mm).

 

Figure 1-5: Somatic embryo-derived complete plantlet, 10 days after being transferred
to hormone-free MS medium (length: 20 mm).

49

 

Figure 1-6: Somatic embryo development into plantlet, and betalain pigment on hypocotyl
after transfer onto hormone-free MS medium.

50

 

'— 100u

 

Figure 1-7: SEM of an early torpedo stage somatic embryo.

 

'—-‘ lOOu

 

Figure 1-8: SEM showing a torpedo stage somatic embryo.

 

 

51

 

 

H—rlOOu
Figure 1-9: SEM showing a late cotyledonary stage somatic embryo.

 

 

 

 

 

 

100u
Figure 1-10: SEM showing an embryo with radicle development fi'om one polar.

 

52

 

.——. 100u

Figure 1-11: SEM showing a torpedo stage of somatic embryo attached to the callus.

 

 

 

 

r—< 100u
Figure l-12: SEM showing a somatic embryo with cotyledons and a little swollen
hypocotyl.

 

 

53

 

 

r—-' 100u

Figure 1-13: SEM showing an abnormal embryo with swollen a swollen hypocotyl.

 

 

 

-—< 100u

Figure 1-14: SEM showing trichomes only occurred on the cotyledonary surface of
embryo.

 

 

 

54

 

~—————~ 100u

Figure 1-15: SEM showing the magnification of partially area fi'om Figure 1-14.
The trichomes appeared on the cotyledons of the somatic embryo.

 

 

 

 

100u
Figure 1-16: SEM showing a somatic embryo with trichomes on the cotyledonary surf ace.

 

 

55

 

Figure 1-17: Encapsulation of somatic embryos. The diameter of each gel bead is about
4-5 mm.

CHAPTER 2
MEDIA AND GENOTYPE EFFECTS ON
SOMATIC EMBRYOGENESIS FROM SUGARBEET CALLUS.
ABSTRACT

Suspension platings of two sugarbeet genotypes, REL-l and REL-2, were used to
determine the efl‘ects on somatic embryo production under varied combinations of abscisic
acid (ABA) with the gowth regulators l-naphthalene acetic acid (NAA), 6-benzyladenine
(BA), triiodobenzoic acid (TIBA), 2,4-dichloroacetic acid (2,4-D), difl‘erent sucrose
concentrations, or difl’erent sole nitrogen sources with the goal of improving somatic
embryo production. Higher BA concentration was reduced embryo numbers but increased
shoot regeneration for both clones. At some BA levels, the presence of ABA improved
somatic embryo and/or shoot numbers up to twenty-fold. TIBA in the range of 0.1-3.0
mg/L had a promotive efl‘ect on somatic embryo production only in combination with
ABA 2,4-D was inefl’ective in improving somatic embryo numbers. Three and five
percent sucrose were superior to one, seven, and nine percent for promoting somatic
embryo production. At some concentrations, urea and glutamine as sole nitrogen sources,
as well as NAA, stimulated geater embryo production over the control, but only in REL-
1, which in general was a much less prolific somatic embryo and shoot producer than
REL-2, one of its progeny. Moreover, ABA at certain concentrations consistently
improved embryo production in all experiments, and was observed to stimulate shoot

production. The range of embryo production was more than thirty-five

56

57

fold between genotypes (REL-2 and REL-1), whereas the range of gowth regulator

efl‘ects was no geater than ten-fold. Use of these clones for transgenic efl‘orts, somatic

cell selection or synthetic seeds should be facilitated by this research.

INTRODUCTION

Plant tissue culture is playing an important part in plant biotechnology directly for mass
clonal propagation and indirectly as a central tool for somatic cell selection or genetic
engineering, in plant modification and improvement. In plant tissue culture, somatic
embryos have most often been produced fi'om callus or suspension culture cells under
appropriate culture conditions, with gowth regulators or genotype dependency being
common. Somatic embryogenesis was first reported in 1958 with carrot (Steward et al.,
1958). Since then, the number of species producing somatic embryos has rapidly
increased. The list is well diversified and includes field crops, vegetables, omamentals and
trees. This success is attributed to a number of factors, such as the search for amenable
genotypes, rapid communication of successful media, common patterns of media induction
of embryogenesis, and a better understanding of the embryo developmental process.
Despite the lengthy period of development of these procedures, somatic embryogenesis is
still a poorly understood phenomenon for most crops. Further research is required to fully
understand and control its use.

Plant gowth regulators play a very important role in eliciting organogenesis or
embryogenesis fiom callus or suspensions. An auxin is usually required in the medium for
induction of somatic embryogenesis and maintenance of proliferative gowth (Ammirato

and Steward, 1971; Vasil et al., 1982). Although 2,4-D was more efi’ective in promoting

58

embryogenesis compared to other auxin sources in some species, such as barley or white
clover (Ziauddin and Kasha, 1990; Parrott, 1991), NAA was a more usefirl auxin for
production of somatic embryos that converted into normal plantlets in asparagus (Levi and
Sink, 1991) and pecan (Rodriguez and Wetzstein, 1994).

Cytokinin is often required for induction of shoot organogenesis, but is not required for
initiation of sOmatic embryogenesis (Ammirato, 1983). Sometimes, cytokinin in
combination with auxin were essential for gowth and induction of somatic embryogenesis
as in carrot cell suspensions (Fujimura and Komanine, 1980). Levi and Sink (1991)
reported that the combination of auxin (NAA or 2,4-D) and cytokinin (kinetin)
dramatically afl‘ected the fi'equency of somatic embryos.

ABA has distinct effects on morphogenesis (Ammirato, 1974 ; 1983). ABA, at the
appropriate concentrations, can inhibit precocious germination and abnormal development
in alfalfa (Fujii et al., 1990), as well as foster normal maturation and conversion in
rapeswd (Finkelstein et al., 1985) and Lin'odenrb‘on tuliptfera (Merkle et al., 1990). ABA
and osmoticum (such as sucrose) can promote maturation of embryos in pea (Barrett and
Clark, 1989; Misra et al., 1993). Xu et al. (1990) reported that precocious germination of
developing alfalfa embryos could be suppressed by ABA and high osmotic potential in.
culture. However, an osmoticum did raise the endogenous ABA level in maize (Rivin and
Grudt, 1991), and then the increased ABA may further promote the induction of
embryogenesis. Besides, ABA also enhanced the conversion of somatic embryos into
plantlets in asparagus (Li and Wolyn, 1995; Li and Wolyn, 1996).

Response in tissue culture inducing somatic embryogenesis varies greatly with

genotype, e.g., soybean (Bailey et al., 1993), watermelon (Compton and Gary, 1993), and

59
asparagus (Delbreil and Jullien, 1994). Thus, somatic embryogenesis and regeneration
ability could probably be increased in vitro by selecting highly responsive genotypes and
subsequent breeding. In alfalfa, Brown and Atanassov (1985) found that 50 of 76 cultivars
failed to produce somatic embryos. Li and Wolyn (1996) reported that only three of six
asparagus genotypes produced somatic embryos.

Somatic embryos in sugarbeet callus or suspension cultures have been reported
anecdotally by a number of authors (Atanassov, 1976; Tetu et al., 1987; Freytag et al.,
1988; Doley and Saunders, 1989; Kubalikova, 1990; Tenning et al., 1992; D’Halluin et al.,
1992). Tetu et al. (1987) concluded that multiple hormonal sequences in the medium were
necessary for the induction and development of somatic embryos fiom callus. In contrast,

Doley and Saunders (1989) reported the simple production and partial germination of

somatic embryos fi’om leaf-disc hormone-autonomous callus of a fodder beet cultivar
without the use of gowth regulators. Tsai and Saunders (1995) extended this work to
sugarbeet biotech clone REL-1 by demonstrating that somatic embryos, albeit in low
fiequency (one per ml of plated suspension), could be recovered from callus after plating
suspensions gown with Murashige-Skoog (MS) (1962) medium plus 1 m BA onto
MS hormone-flee medium. This same work determined that catain concentrations of
ABA in the plating medium increased the somatic embryo yield of REL-l as much as
fifteen-fold.

In order to increase somatic embryo number, two sugarbeet genotypes (REL-1 and
REL-2) were used to measure the level of somatic embryo production from suspensions
plated onto MS medium with combinations of ABA with difl’erent concentrations of

gowth regulators, sucrose, or difl‘erent sole nitrogen sources in this studies.

60

MATERIALS AND METHODS
W

The experiments were performed with several genotypes of sugarbeet (Beta vulgaris
L.). REL-1 (J.W. Saunders, East Lansing, MI.) is a self-fertile diploid sugarbeet clone
bred for ease of tissue culture manipulations. It exhibits a high frequency of leaf disc
callusing, shoot regeneration from hormone autonomous callus, dispersed suspension
cultures and resistance to shoot vitreousness. REL-1 has been used for the recovery of
monogenic dominant sulfonylurea herbicide resistance by somatic cell selection (Saunders
et al., 1992). REL-2 (also known as LTR-41) is an F1 hybrid clone between EL 45/2-108
(Doley, 1990) and REL-l. Three additional clones were tested in one experiment: Clone
6926cms12 (fiom SP6926-01) (G. Coe, Beltsville, 1WD), Clone GWK-R, an individual
plant fiom the mating of two somatic-embryo-derived plants fi'om the fodder beet cultivar
Garton’s White Knight, and Clone 6822-08 fiom SP6822 (J .W. Sarmders, East Lansing,
MI).

Both REL-1 and REL-2 have been maintained in shoot culture (Saunders,l982). LTR-
41hasbeenrdeasedasREL-2sothatargarbeetresearcherscmuseitsarpaior
embryogenic and shoot regeneration abilities for application in biotechnology.

QM; media
The culture media contained MS mineral salts (Murashige and Skoog, 1962),
supplemented with 100 mg/L myo-inositol, 1.0 mg/L thiamine‘HCl, 0.5 mg/L nicotinic

acid, 0.5 mg/L pyridoxine‘ HCl, and 30 g/L sucrose. Media used for leaf disc callusing

61

were gelled with 9 g/L Difco Bacto agar, whereas media used in plating out were gelled
with 3.5 g/L phytagel. The gowth regulators used were NAA (0.3-10 mg/L), BA (0.1-1.0
mg/L), TIBA (0.1-10 mg/L), 2,4-D (0.1-1.0 mg/L), and/or ABA (0.02-3.0 mg/L). The pH
was adjusted to 5.95 with KOH or HCl before autoclaved (15 psi, 121 °C, 20 min). ABA
was filter-sterilized and added into previously autoclaved and partially cooled media as
were sole nitrogen sources. Nitrate (N 03), ammonium (NI-L) urea, glutamine (GLN),
glutamate (GLU), proline (PRO), glycine betaine (BET) and choline (CHO) were tested as
sole nitrogen source at 60 mM atomic nitrogen. KNO; (20 mM) and NHaNog (40 mM)
were used as the standard MS nitrogen Nitrogen-fies (MS-N) served as the control. NH4
was provided as the chloride salt and was accompanied by half that concentration of
succinic acid.

Culture vessels were 125 mL Erlenmeyer flasks for suspension-culture or 20 x 100 mm
Falcon disposable plastic Petri plates for callus initiation and suspension plate-out.
Medium volume per vessel was 35 ml. Flasks were closed with foam caps and alumimrrn
foil. Petri plates were sealed with one layer of Parafilm

Callus was initiated fiom leaf discs (8 mm diameter) cut fiom partially expanded leaves
of geenhouse gown plants. Leaves were sterilized for twemy mimrtes with a dilute (1:5
v/v) commercial (5.25 % w/v) sodium hypochlorite (NaOCl) bleach containing 100 mg/L
sodium lauryl sulfate detergent followed by five rinses in sterile distilled water. Leaf discs
were placed on MS medium with 1 mg/L BA (Saunders et al., 1992) in Petri plates
incubatedat30°Cinthedark, onediscperplateof35mLmedium Calluswasfirst
observed after one month, and after another month (fi'om REL-1); or after 2-3 weeks

(from REL-2), 2 to 3 g of leaf-disc callus was transferred to liquid hormone-fies MS

62

medium in flasks for gowth at 21:2 °C in the dark. However, REL-2 required only half a
month before callus was taken for suspension culture because it gew faster. The
suspension cultures were shaken on rotary shakers at 125 rpm to aerate the cultures and to
reduce cell cluster size.

Suspension cultures were subcultured with hormone-free MS medium every 5-7 days.
After 10 to 14 days, suspensions used as inoculum for platings were pushed through a
stainless steel sieve with 830 um openings. For experiments with difi‘erent nitrogen
sources or sucrose levels, sieved suspensions were washed with nitrogen-flee or sucrose-
fi'ee liquid medium, respectively. Sieved suspension cell clusters were plated on MS media
with the indicated combinations of gowth regulators. Plates were inoculated with 1 ml
(approximately 0.1 g fiesh weight) of suspension by plating out with disposable plastic
Pasteur pipettes. Afier inoculation, plates were sealed with Parafilm strips and incubated
in dim light (less than 5 umol m'zs") fi'om fluorescent lamps (Philips cool white
econowatt) at 25 °C. Experiments were terminated after 4-5 weeks. Minimum size for an
embryo to be counted was 0.5 mm.

The number of somatic embryos and shoots, as well as fresh weight of somatic embryos
and callus were statistically analyzed. The two factor randomized complete block design
with 6-10 replications per treatment was used, and experiments were performed with
similar results 2 or 3 times each. The analysis of variance was performed using MSTAT.

The least significant difi'erence (LSD, P=0.05) was used to separate treatment mean.

63

RESULTS AND DISCUSSION

Initiation of leaf disc callus and susp_ension cultures

In preliminary experiments, clone REL-2 was developed primarily as an embryogenic
genotype, using somatic embryo production on primary leaf disc callus initiating on
hormone-flee medium as evaluation criterion (Figure 2-1). Callus initiation time was 2
months (twice as long compared to the response on MS medium with 1 mg/L BA), and
afier3 months, aboutathird ofsuchcallihad aroughfirzzywhiteareaofwhatappeared
to be somatic embryos or white embryogenic tissue (Figure 2-2).

Whenleafdisccalluswasinitiated onMS mediumwith l mg/LBAindarknessat30
°C, shoot regeneration was ultimately seen with genotypes REL-1 (Figure 2-3) and REL-2
(Figure 2-4). REL-1 leaf disc callus first appeared after about one month, and after
another month, the fiiable callus was transferred to liquid hormone-flee MS medium for
suspension culture (Figure 2-5). Most REL-2 leaf disc callus underwent shoot
regeneration quickly after callus was observed. Therefore, REL-2 only required two
weeks before callus was transferred to hormone-free MS liquid medium for suspension
culture since its callus quickly changed color to brown after a short time. These

suspension culture establishment procedures were applied to the following experiments.

d ' n ' ' ABA and NAA
With genotype REL-1, a low frequency of somatic embryogenesis, less than one
embryo per petri plate, was seen following plating-out of subsequent suspension cultures

on hormone-flee MS medium. Suspensions of REL-l plated onto media with

64

combinations of NAA and ABA produced low numbers of embryos (Figure 2-6), but ABA
at 0.1 or 0.3 mg/L in the medium increased the number of somatic embryos per plate up to
nine fold (Figure 2-7). Nearly one embryo per plate was found on the basal medium
without containing NAA and ABA Maximal production, more than six embryos per plate,
occurred on the medium with 1.0 mg/L NAA and 0.1 mg/L ABA ABA is well known for
its positive efl‘ects in somatic embryogenesis, where it normalizes development and inhibits
precocious germination (Ammirato, 1974, 1983).

For genotype 6926cm312, a very low number of somatic embryos was produced when
similar combinations of NAA and ABA were used (Figure 2-8). This genotype produced
embryos only with 1 mg/L NAA + 0.1 mg/L ABA (one embryo per plate) or 3 mg/L NAA
+ 0.1 mg/L ABA (0.3 embryo per plate).

For genotype GWK-R and genotype 6822-08, no somatic embryos were obtained in
this system when similar combinations of NAA and ABA were used.

Wrth genotype REL-2, much higher fiequency of somatic embryogenesis (Figure 2-9)
was seen, with nearly 40 embryos per plate on basal medium (Figure 2-10). The inclusion
of 0.1 or 0.3 mg/L ABA alone in the plate out medium sigrificantly increased the
production of somatic embryos (Figure 2-11, 2-12). Maximum average somatic embryo
yield observed was 77 per ml of suspension plated out on the MS medium containing 0.3
mg/LNAAand0.1mg/LABA Ontheotherhand, highlevels of ABA(3 mg/L) or
NAA (3 and 10 mg/L) decreased the combined fi'esh weight ofsomatic embryos and callus
per plate (Figure 2-13).

Actually, difl‘erent genotypes strongly afl‘ected somatic embryogenesis and the mrmber

of somatic embryos, as least as much as any gowth regulator combination did. Similarly,

65
other reports showed how difl‘erent genotypes affect somatic embryogenesis such as with
gape (Emershad and Rarnming, 1994), and radish (Takahata et al., 1996). Genotype
along with auxin had a sigrificant effect on the fi'equency of somatic embryogenesis in

soybean (Parrot et al., 1989) and in chickpea (Eapen and George, 1994).

Induction of somatic embryos with ABA and 2,4-D
Concentrations of 2,4-D from 0.1 to 1.0 mg/L with or without ABA did not strongly

afi‘ect the frequencies of somatic embryogenesis with REL-2 (Figure 2-14). Media
containing ABA at 0.1 mg/L had significantly higher numbers of somatic embryos than did
those with 0, 0.3 or 1.0 mg/L. Thus, ABA was far more efi‘ective for promoting somatic
embryos than was 2,4-D. In some species, 2,4-D was more efl‘ective in promoting somatic
embryogenesis than were other auxins (NAA or IAA). For instance, 2,4-D was superior to
NAA or IAA for embryogenic callus initiation or maintenance in barley (Ziauddin and
Kasha, 1990). Nevertheless, Rodriguez and Wetzstein (1994) reported that more pecan
embryos were induced by 2.4-D, but NAA was more efi'ective in inducing somatic
embryos morphologically resembling zygotic embryos and their subsequent conversion

into plantlets.

Induction of somatic embrlos and shoots with ABA gd BA
Combinations of ABA with BA were evaluated for efl’ects on production of somatic

embryos and shoots in genotypes REL-1 and REL-2. A low frequency of somatic
embryogenesis (not higher than six embryos per mL of plated suspension) was seen

following plating onto media containing various combinations of BA and ABA fiom

66
genotype REL-1 (Figure 2-15). However, the yield of shoots obtained through
organogenesis was progessively higher as concentrations of both BA and ABA increased
(Figure 2-16). Approximately 68 shoots per plate were induced with 1.0 mg/L BA and 2
mg/L ABA (Figure 2-17).

With genotype REL-2, a similar pattern of lower embryo and higher shoot numbers
with progessively higher BA concentration was observed following plating-out of
suspension (Figure 2-18). Anyway, the yields of somatic embryos and shoots were
considerably higher than with REL-1. REL-2 produced nine times more embryos in the
absence of BA when averaged over ABA concentrations, and 3 .5 and 2 times more shoots
at 0.3 and 1.0 mg/L BA, respectively. Moderate ABA concentrations (0.02 or 0.2 mg/L)
stimulated embryo production, and the efl‘ect was significant at 0 and 0.1 mg/L BA BA
inhibited embryogenesis at all concentrations, regardless of the presence or absence of
ABA ABA significantly stimulated shoot production in combination with 0.3 or 1.0 mg/L
BA (Figure 219).

However, ABA had a strong positive influence on both somatic embryo and shoot
production in an interactive manner: at least one of the three ABA concentrations
enhanced either somatic embryo or shoot production at the two best BA levels,
respectively, with both genotypes REL-1 and REL-2 (Figure 2-15, 2-18). With the less
productive clone REL-l, progessive increases in shoot number were evident with
increasing initial ABA concentrations at the two highest starting BA levels, to the point
where at the combination of highest levels of both ABA and BA, shoot production
reachedanintensitymatchingthatseeninthe absence ofABAinthestartingmediumwith

REL-2.

67

Induction 9f somatic embggos and shoots with ABA and TIBA in the pmce of 1.0
m BA

Combinations of TIBA and ABA were tested at 1.0 mg/L BA, a level very conducive to
shoot production but not very permissive of somatic embryo production TIBA at 10
mg/L severely inhibited both somatic embryo and shoot formation in both REL-1 and
REL-2 genotypes (Figure 2-20, 2-21). Overall, TIBA in the range of 0.1-3.0 mg/L had a
promotive efi‘ect on somatic embryo production only in combination with ABA, to a minor
degee, and more so for REL-1 than for REL-2. By itself TIBA had no promotive efl‘ect
on shoot formation with either genotype, but for REL-1 in the TIBA concentration range
0.1-3.0 mg/L (4 treatment levels), it interacted positively with ABA at the 2.0 mg/L
treatment level to double or even triple shoot numbers per plate (Figure 2-20). From the
ABA perspective, genotype REL-1 was much more responsive, and this was seen as an

interactive efl‘ect on shoot production.

Indugg'on 9f somatic gmbrygs with ABA and sucrose
With genotype REL-2, the influence of sucrose concentration in the plating medium on

somatic embryo production was examined with and without ABA at 0.2 mg/L. Increasing
the sucrose concentration fiom l to 3 or 5% significantly increased embryo production, in
the absence of ABA (Figure 2-22). ABA had no significant efl‘ect at 1, 7, and 9% sucrose,
but approximately doubled embryo production with 3 and 5% sucrose. The positive efi'ect
of sucrose concentration on somatic embryo production has also been documented by

Gray et al. (1993) with Cucumis melo. Komura et al. (1990) reported that 2-3 % sucrose

68

was the optimum concentration for embryogenic callus induction in asparagus, but somatic

embryos only developed with 5 % sucrose.

Induction of somatic embgos with ABA and sole nitrogm sgurgs
Combinations of sole nitrogen sources and ABA (0.02, 0.2, 2 mg/L) as the only gowth

regulator were examined in the plating medium for both genotypes, REL-1 and REL-2.
With REL-l, only urea and GLN dramatically increased embryo production on the media
containing 2.0 and 0.2 mg/L ABA, respectively, over the MS nitrogen mix control (Figure
2-23). Embryo production for most nitrogen sources was geater for at least one of the
ABA concentrations than in its absence. Media containing no nitrogen, GLU, BET, or
CHO formed almost no embryos. In general, shoot formation for each nitrogen source
reflected embryo production Very few embryos or shoots were produced on BET, CHO,
or in the absence of nitrogen.

Maximum embryo production was four times as high with REL-2 than with REL-l, and
no nitrogen source gave a sigiificantly higher production than the MS nitrogen mix
control (Figure 2-24). Only urea, GLN and the MS nitrogen mix produced considerable
numbers of somatic embryos. In addition, with the more embryogenic genotype REL-2,
shoot production did not reflect embryo production when nitrogen sources were
compared to genotype REL-1. BET, CH0, and the nitrogen-fies medium (MS-N) were
unproductive of embryos or shoots.

In other crops, supplemental PRO stimulated embryogenic callus in carrot (Nuti-Ronchi
et al., 1984), maize (Armstrong and Green, 1985) and Agrostrs alba (Shetty and Asano,

1991). Supplemental GLN promoted somatic embryogenesis in suspension cultures of

69
Gosspium klotzschianum (Price and Smith, 1979). Moreover, supplementation with GLN
also increased somatic embryo size and increase storage protein in alfalfa (Lai and

McKersie, 1994).

Development pf gmatic embryos
During the first two weeks following plating-out, white or light yellow callus

proliferated. From the third to the sixth week, various late stages of somatic embryos,
fiom torpedo to mature opaque white embryos with cotyledons, were clearly
distinguishable at the callus surface (Figure 2-25). The simultaneous occurrence of
somatic embryos of difl‘erent lengths (0.5 to 4 mm) indicated that somatic embryogenesis
was not uniform and synchronous. After 40 days, most of the somatic embryos with
cotyledons were around 2 - 3 mm long. Each somatic embryo could be easily separated

from the surrounding callus.

The promotive cfi‘ect of ABA on somatic embryo number as initially reported by Tsai
and Saunders (1995) with REL-1 has been much more extensively confirmed with the
second genotype REL-2. Difl‘erent genotypes and media under varied combinations of
ABA with other gowth regulators (NAA, 2,4-D, BA, TIBA), difl‘erent sucrose
concentrations or difl‘erent sole nitrogen sources strongly afl‘ected somatic embryo
production. Bell et al. (1993) also reported that exogenous ABA promoted somatic
embryo number in orchardgass, and high internal levels of ABA have been implicated in
permitting somatic embryogenesis fi'om leaf explants of Napier gass (Rajasekaran et al.,

1987). The involvement of ABA in regulating the continued normal development of

70

somatic embryos was demonstrated by Ammirato (1974; 1983). The application of ABA
also prevented precocious germination and improved desiccation tolerance in somatic
embryos of alfalfa (Senaratna et al., 1989; Anandarajah and McKersie, 1990). During the
later stages of embryogeny, high osmoticum affected accumulation of storage proteins in
rapeseed embryo development (F inkelstein and Crouch, 1986). ABA might suppress water
uptake to attain the desiccation phase, since desiccation is required to complete embryo
maturation. On the other hand, application of high concentrations of ABA to carrot
somatic embryos caused dedifl‘erentiation to embryogenic callus-like structures (Iida et al.,
1992). Thus, the gowth regulator ABA plays an important role fiom the initiation of
somatic embryogenesis and further development as well as conversion into plantlets.

In this research, sugarbeet genotype REL-2 was first found to have superior somatic
embryogenesis and shoot regeneration abilities. In addition, this research also showed that
exogenous ABA significantly promoted the fi'equencies of somatic embryo and shoot
production from both genotypes (REL-1 and REL-2). Therefore, we can utilize these

clones in firture studies of somatic cell selection, artificial seeds and genetic

transformation.

71

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Figure 2-1: Somatic embryo production on primary leaf disc callus (28 mm) initiating
on hormone-free MS medium after 58 days. (REL-2)

Figure 2—2: Somatic embryos on callus initiated from a leaf disc on hormone-flee MS
medium. About a third of such calli have rough fuzzy white embryogenic
tissue. (REL-2)

 

77

 

Figure 2-4: Leafdisc callus fi'om MS medium with 1 mg/L BA (REL-2 also known
as LTR-41)

78

 

Figure 2-5: F riable callus was transferred to liquid hormone—free MS medium for
suspension culture. (REL-l)

Figure 2-6: Plate out callus with a low number of somatic embryos (opaque white)
on MS medium with 0.25 mg/L NAA and 0.1 mg/L ABA after 29 days.

(REL-1)

79

 

 

 

 

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Figure 2-7; The influence of NAA and ABA on somatic embryo production with

genotype REL-1 after 29 days. Means marked with same letter are not

significantly difi‘erent according to LSD (P<0.05).

80

 

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81

 

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and 0. 3 mg/L ABA after 40 days. (REL-2)

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after 31 days. (REL-2)

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production with genotype REL-1 after 34 days. Means marked with the same

letter are not significantly difi'erent according to LSD (P<0.05).

94

 

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ficantly different according to LSD (P<0.05).

th genotype REL-2 after 34 days. Means marked with the same

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95

 

Figure 2-25: Difi‘erent stages of somatic embryos were clearly distinguishable at the callus
surface. (REL-2)

CHAPTER 3
ENCAPSULATION, GERMINATION, AND CONVERSION OF
SOMATIC EMBRYOS IN SUGARBEET CLONE REL-2
ABSTRACT

Experiments with encapsulation and subsequent germination and conversion of
sugarbeet somatic embryos to plantlets were performed with 2% sodium alginate, in initial
efforts necessary for development of artificial seeds. Factors examined were encapsulated
embryo size, alginate companion solution, cold storage duration, and germination
substrate. Somatic embryo length category (0.5-1.9, 2.0.2.9, 3.0-3.9 mm) did not afl‘ect
germination or conversion rates (87, 89, 87 %, respectively) into complete plantlets on
hormone-flee Murashige-Skoog (MS) medium in the absence of encapsulation. Alginate
companion solutions (either MS medium or H20) had no difi‘erential efi‘ect on germination
rate (100 %) but did difl‘er in converting embryos to plantlets (81 vs. 64 %, respectively).
No loss of conversion ability at 25 °C occurred after 21 days of cold storage at 4 °C of
encapsulated embryos. After 64 days of storage at 4 °C, the germination and conversion at
25 °C of embryos encapsulated with alginate in MS medium had dropped to 70 °/o and 45
%, respectively, with alginate in H20 respective rates were 60 % and 20 %. In addition,
the germination rate in soil plates after 64 days cold storage for alginate capsules in MS
medium or in H20 was 38 % or 25 %, respectively. This initial research showed that
somatic embryos, either nonencapsulated or encapsulated converted into plants at high

frequencies (88% and 81%, respectively) without cold treatment. Cold storage did not

96

97
improve the conversion rate of encapsulated embryos, but did slow their development.
However, this experiment indicated that the nonencapsulated and encapsulated embryos
fi'om clone REL-2 were capable of direct germination after planting on the agar plates and

soil.

98
INTRODUCTION

The concept of using somatic embryos in synthetic seeds was the first proposed by
Murashige (1977). The encapsulation of somatic embryos in a protective coating with
alginate was initially reported by Redenbaugh et al. (1984), whereby alfalfa somatic
embryos were mixed with sodium alginate and dropped into a calcium salt solution to
form calcium alginate capsules. Practical synthetic seed technology would combine the
eficient delivery aspects of seed propagation with elite germplasm conservation and plant
propagule production via in vitro somatic embryogenesis. The advantage of such a
synthetic md system would be the use of conventional seed-handling techniques for
embryo delivery and storage. Somatic embryos would be delivered from tissue culture
through mechanical handling to automated planting. Research on encapsulated embryos
has been reported for several species, and recovery of plants fi’om encapsulated embryos
has been obtained for some crops, e.g., carrot (Redenbaugh et al., 1987a; Li, 1993; Wake
et al., 1995), orchardgrass (Gray et al., 1987), alfalfa (Redenbaugh et al., 1986, 1987b,
1988, 1990; Zhong and Wang, 1989), barley (Datta and Potrylrus, 1989), eggplant (Rao
and Singh, 1991), celery (Kim and Janick, 1987; Li et al., 1990; Nadel et al., 1995), wheat
(Li et al., 1990), and horseradish (Shigeta and Sato, 1994).

The principal limiting problem for synthetic seeds is the dificulty in producing high
quantities of quality somatic embryos, which is critical to the development of complete
plantlets. In general, promoting embryo maturation with abscisic acid (ABA) or cold
treatment did improve the quality of somatic embryos (Fujii et al., 1993). Additionally, the
choice of hydrogel for encapsulation of somatic embryos is crucial. Sodium alginate, the

most widely used hydrogel for encapsulation of embryos, can dissolve easily and remain

99

stable at room temperature (Redenbaugh et al., 1993). The problem afier encapsulation is
maintenance of embryo viability and capsule integrity, as the conversion rate may fall
dramatically after short or long-term preservation. Thus, cold storage was tested in some
systems (Datta and Potrykus, 1989; Li et al., 1990; Arrillaga et al. 1994). Cold treatments
have improved the conversion rate from somatic embryos of Vitz’s (Rajasekaran et al.,
1982). Kitto and Janick (1985) reported cold treatment hardened carrot embryos and
promoted better survival alter embryo desiccation. For alfalfa somatic embryos, cold
exposure for up to 12 weeks did not decrease the conversion rate in soil in a growth
chamber, with the conversion rate of the alfalfa embryos in soil still more than 40 % alter
18 weeks of cold storage (Fujii et al., 1993). Nevertheless, cold storage could slow
development, or biochemical and metabolic changes in the embryos.

A fairly prolific system for primary somatic embryogenesis has been established for
sugarbeet clone REL-2 (Chapter 2). This system produces large enough numbers of
somatic embryos for experiments on their utilization The purposes of this research were
to (I) examine the germination and conversion rates of difierent length categories of
somatic embryos of sugarbeet clone REL-2, (2) and to examine the germination and
conversion rates of somatic embryos encapsulated with alginate in MS medium or in HzO

(i.e., without nutrients) afier difi‘erent periods of cold storage at 4 °C.

1 00
MATERIALS AND METHODS
Experiments were performed with diploid sugarbeet (Beta vulgaris L.) clone REL-2,

released to the public in 1996. REL-2 clone had been developed specifically for superior
embryogenesis and shoot regeneration, annual biotech clone REL-l (Tsai and Saunders,
1995) being one of its parents. Clone REL-2 was maintained in shoot culture (Saunders,
1982) and is available upon request, either as in vitro shoots, whole plants, or S; seeds.

The basic culture medium contained MS mineral salts (Murashige and Skoog, 1962),
100 mg/L myo-inositol, 1.0 mg/L thiamine’HCl, 0.5 mg/L nicotinic acid, 0.5 mg/L
pyridoxine’HCl, and 30 g/L sucrose. Media used for leaf disc callusing were gelled with 9
g/L Difco Bacto agar, whereas media used in plating were gelled with 3.5 g/L phytagel. The
pH was adjusted to 5.95 with KOH prior to autoclaving (15 psi, 121°C, 20 min). ABA was
filter-sterilized and added into previously autoclaved and partially cooled media. Culture
vessels were 125 mL Erlenmeyer flasks or 20 x 100 mm Falcon disposable plastic Petri
plates. Medium volume per vessel was 35 mL. Flasks were closed with foam caps and
aluminum foil. Petri plates were sealed with one layer of Parafilm.
Induction of somatic embryos

Callus was initiated fiom leaf discs (8 mm diameter) fiom partially expanded leaves of
greenhouse-grown plants on MS medium with 1.0 mg/L BA (Saunders et al., 1992) in Petri
platesat30°Cinthedark. Calluswasfirstseenafieronemonth. Thenafier 15 days, 2to 3
g of leaf-disc callus (Figure 3-1) was transferred to hormone-free liquid MS medium in 125
mL Erlenmeyer flasks for growth at 2112 °C in the dark. Suspension cultures were shaken

on rotary shakers at 125 rpm to aerate the cultures and to reduce cell cluster size.

1 01

After five to seven days, suspension cultures were subcultured with liquid hormone-fires
MS medium for another five to seven days by pouring fresh medium into the old suspension
flask, mixing, then dividing the suspension evenly into two flasks. Suspensions used as
inoculum for platings were pushed through a stainless steel sieve with 830 um openings.
Sieved suspension cell clusters were plated on MS medium with 0.3 mg/L NAA and 0.1
mg/L ABA Each Petri plate received 1 ml of suspension preparation containing about 0.1 g
fi'esh weight of cells, and was incubated under dim light (less than 5 umol m'zs")fi'om
fluorescent lamps (Philips cool white econowatt) at 25 °C.
Germination and conversion of somatic embgos

Somatic embryos from three length categories (0.5-1.9 mm, 2.0-2.9 mm, 3.0—3.9 mm)
were transferred onto hormone-free MS agar medium or soil, then placed under fluorescent
lamps (20-50 umol m'zs'l) at 25 °C. Germination and conversion rates were determined for
each length category. Germination was defined as development into cotyledons and/or
radicles, conversion as germination and development into plantlets with shoots and roots.
Enc_apsulation of somatic embryos
Somatic embryos (2-3 mm) were immersed in 2 % sodium alginate with either hormone-flee
MS medium containing 3% sucrose or with H20. A pipette was used to suck up single
embryos with the alginate, which were then dropped into 50 mM CaClflHzO and stirred
(80 rpm) for 20 min, thus forming sodium alginate beads of a size (4-5 mm) large enough to
cover the entire somatic embryo. The beads were rinsed with water and then directly placed
on the agar plates or in soil in plates in the light at 25 °C, or stored in darkness at 4 °C.

After storage for O, 9, 21, 30, 41, 50, or 64 days, the somatic embryos encapsulated with

alginate in MS medium or H20 were transferred to agar plates with MS medium or to Petri

1 02
plates with moistened sterile soil (Baccto professional planting mix, Michigan Peat Co,
Houston, TX, USA), then the germination and/or conversion rates were determined in the

light during the next 14 days. Data analysis was based on a chi-square at p50.05.

RESULTS AND DISCUSSION

Induction of somatic embgzos

According to previous work (chapter 2), a high frequency of somatic embryogenesis was
seen following plating-out of suspension cultures of REL-2 on favorable media. The
inclusion of 0.3 mg/L NAA and 0.1 mg/L ABA in the plate-out medium significantly
increased the production of somatic embryos when compared to the plate-out medium
without hormones. Maximum average yield was 77 somatic embryos per ml of suspension
plated out afier 31 days (Figure 3-2). Embryos of difi‘erent lengths (0.5 mm to 4 mm) were
clearly present simultaneously at the callus surface (Figure 3-3). This indicated that somatic
embryogenesis was not synchronous in this system. Some somatic embryos (as in Figure 3-
4) would directly develop into complete plantlets without transfer. Most often, afier transfer
of the somatic embryo onto hormone-free MS medium, its opposing ends gradually
developed into shoots and roots simultaneously to form seedling-like plantlets (Figure 3-5,
3-6).

Sometimes, a very low proportion of somatic embryos experienced some secondary
embryogenesis (Figure 3-7). In sugarbeet, Tenning et al. (1992) reported that such

secondary somatic embryos were produced from the primary embryos.

1 03

In general, the different developmental stages of somatic embryos also correspond to
difi‘erent sizes of somatic embryos. The examination of the germination and conversion
fi'equency for three relatively advanced length categories of somatic embryos which were
placed on hormone-free MS agar plates (Table 3-1) indicated that there was no significant
difi‘erence among germination rates (98 %, 99 %, 100 %) or conversion rates (87 %, 89 %,
87 %) when embryos from three length categories (total range = 0.5 to 3.9 mm) were
placed on agar plates. The difi‘erence between germination rates and conversion rates into
complete plantlets was 10-13% (Table 3-1) because of subsequent callusing or the
development of abnormal advanced embryos and plantlets. Nevertheless, the germination
or conversion rates might be diEerenfially afi‘ected at difl’erent stages of embryonic
development with other genotypes or species. For instance, the smallest length category
(0.5-1.9 mm) had more dificulty germinating and converting into complete plantlets than
did the other embryo sizes (2.0-2.9 mm; 3.0—3.9 mm) in earlier experiments with genotype
REL-1 (Tsai and Saunders, 1995). Furthermore, with REL-2, respective germination and

conversion rates were similar for nonencapsulated embryos placed on the MS medium agar

plates or on the soil plates (Table 3-2).

Encapsulation of somatic embryos

We utilized isolated somatic embryos (Figure 3-8) for encapsulation to initiate study of
the possibility of making artificial seeds (Figure 3-9). The diameter of each gel bead in
diameter was approximately 4-5 mm (Figure 3-10). Somatic embryos encapsulated with 2%
sodium alginate in MS medium demonstrated 100 % germination and 81 % conversion in
the absence of any storage; 2 % sodium alginate in H20 (i.e., without nutrients) resulted in

100 % germination and 64 % conversion when placed on agar plates with MS medium

1 04

without any storage (Table 3-3). Several reports have indicated that encapsulation of
somatic embryos inhibited the conversion rates as compared to nonencapsulated somatic
embryos. For instance, Rao and Singh (1991) reported a lower conversion rate for
encapsulated (49.7%) in comparison with unencapsulated somatic embryos of Solanum
melongena L.. A contrasting pattern was apparently observed by Shigeta et al. (1990) who
noted a significantly greater conversion fiequency in carrot with 54 % (nonencapsulated)
and 74 % (encapsulated). Datta et al. (1989) reported that alginate-encapsulated somatic
embryos of barley performed better than nonencapsulated embryos, perhaps by limiting
oxygen access. In any case, the condition of the embryos entering encapsulation, as well as
the actual encapsulation method or material, could account for such response difi‘erences.

At 25 °C encapsulated embryos started germinating quickly (Figure 3-11, 3-12, 3-13)
after being placed on MS agar plates. Some encapsulated embryos developed into normal
plantlets, and produced betalain pigment on hypocotyls (Figure 3-12, 3-13). In addition,
some encapsulated embryos germinated out of the alginate, and then developed cotyledons
and radicle nearly simultaneously (Figure 3-14). Storage durations of 0-21 days at 4 °C did
not significantly afi'ect response. In alfalfa, conversion of alginate-encapsulated somatic
embryos was inhibited by exposure to 4 °C for only 1 week (Janick et al., 1989;
Redenbaugh et al., 1991). In contrast, Anillaga et al. (1994) reported that a 15-day cold
treatment significantly increased the conversion rate for nonencapsulated black locust
embryos (from 70 % increasing to 95 %) and for encapsulated embryos (fi’om 41 %
increasing to 80 %), but longer periods of cold storage drastically reduced the conversion
rate. In this sugarbeet study, after 64 days of storage at 4 °C, the germination and

conversion rate of encapsulated embryos with MS medium was 70 % and 4S %,

1 05

respectively; compared with 60% and 20% for beads without nutrients (alginate in water)
(Table 3-3). The longer the encapsulated embryos were stored, the lower the conversion
rate. The lowest conversion frequency was found in the encapsulated coats with alginate in
water when compared to the encapsulated coats with alginate in hormone-flee MS (medium
A few somatic embryos germinated out of encapsulation in an unbalanced manner (Figure
3-15) after 41 days of cold storage. However, a very low percentage of the encapsulated
embryos with alginate in MS medium still were viable afier 191 days of storage at 4 °C, but
most germinated directly out of the beads and became brown (data not shown). Datta and
Potrykus (1989) reported that they succeeded in preserving encapsulated barley embryos for
six months at cold temperatures (4°C), and at the end of this period achieved 37 %
germination. In black spruce, the germination fiequency showed a sharp reduction afier one
month of cold storage whether encapsulated or not (Lulsdorf et al., 1993). In addition,
Gupta and Durzan (1987) found that encapsulated somatic embryos of loblolly pine did not
convert into plantlets afier storage at 4 °C for 4 months. In all such studies, the quality of
the somatic embryos would be critical. Li et al. (1990) noted that with carrot, the vigor
index, dehydrogenase activity, and respiration rate all decreased as storage duration
increased. An ideal system would delay the further growth of somatic embryos, preserve
the encapsulated embryos for high viability, and then further maintain or improve the
conversion rate.

VVrthout cold treatment, 53 % of encapsulated embryos fi'om sugarbeet clone REL-2
germinated in soil (Table 3-4). Zhang et al. (1990) reported that the germination rate of
encapsulated carrot embryos was as high as 87 % in nonsterile vermiculite when mrtrient

medium and a preservative were included in the alginate capsule. Fujii et al. ( 1987)

1 06
observed that the plant recovery rates were unacceptably low if the hydrated encapsulated
embryos of some crop species (alfalfa, carrot, celery, and cauliflower) were planted in soil.
In another experiment, the cold treated encapsulated embryos were directly
transferred onto the soil mix in Petri plates to examine only the germination rate (Table 3-
4). There was no significant difi‘erence between germination rates with the beads containing
MS medium and those without nutrients (i.e., H20) with 50 or fewer days of cold storage.
After 64 days of storage, the germination rate was only 38 % and 25 %, respectively, when
beads coated with 2% alginate in MS medium or in H20 were placed in soil. The
germination rates were dramatically lower when encapsulated embryos were planted on soil
compared to MS medium agar plates (Table 3-3, 3-4).
In conclusion, aside fiom the quality of somatic embryos, the optimized capsule
supportive complex, the capsule companion solution and the planting medium all appear to
afi‘ect the germination and conversion rates, as well as storability. In order to establish a

good system for encapsulating somatic embryos, we need to consider all of these factors in

the firture experiments.

107

REFERENCES

Arrillaga, I., 1.]. Tobolski, and SA Merkle. 1994. Advances in somatic
embryogenesis and plant production of black locust. Plant Cell Rep.
13: 171-175.

Datta, SK. and I. Potrykus. 1989. Artificial seeds in barley: encapsula-
tion of microspore derived embryos. Theor. Appl. Genet. 77: 820-824.

Fujii, J.AA, D. Slade, and K. Redenbaugh. 1993. Planting artificial
seeds and somatic embryos. In Synseeds. K. Redenbaugh. (ed.). CRC
Press, California. pp 183 -202.

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110

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Table 3-1: Efi‘ect of length category on the proportions of somatic embryos germinating

111

and converting into complete plantlets in the absence of encapsulation.

 

 

 

 

Length of somatic embryos(mm) Germination rate Conversion rate
‘ 0.5 - 1.9 98%(82/84) a 87%(73/84) a

2.0 - 2.9 99%(95/96) a 89%(85/96) a

3.0 - 3.9 100%(38/38) a 87%(33/38) a

 

 

 

Means within columns followed by the same letters were not significantly
difi‘erent based on a chi-square at p50.05.

Table 3-2: Germination and conversion rates of nonencapsulated somatic embryos

on the MS medium or the soil without cold storage.

 

 

 

 

 

MS medium (agar 9 g/L) germination rate 100% (16/16) NS
conversion rate 88% (14/ 16) NS

Soil germination rate 100% (10/ 10) NS
conversion rate 90% (9/10) NS

 

 

NS indicated that germination and conversion rates on MS medium
or soil were not significantly difl‘erent based on a chi-square at p_<_0.05.

112

Table 3-3: Comparison of germination and conversion rates on MS medium of somatic
embryos alginate-encapsulated with either MS medium or H20 afier storage

 

 

 

 

 

 

 

 

 

at 4 °C.
Days of storage Germination response Conversion response
at 4 °C alginate encapsulated with alginate encapsulated with
MS medium H2O MS medium H10

0 100%(16/16)a 100%(14/14)a 81%(13/16)a 64%(9/14)aeflr

9 95%(19/20)ac 85%(17/20)ad 75%(15/20)ac 65%(13/20)aefi
21 95%(19/20)ac 75%(15/20)bcde 60%(12/20)ad 55%(1 1/20)aefi
30 85%(17/20)ad 80%(16/20)ae 40%(8/20)bde 40%(8/20)bde
41 80%(16/20)ae 75%(15/20)bcde 45%(9/20)bcdf 45%(9/20)bcdf
50 80%(16/20)ae 80%(16/20)ae 50%(10/20)aefg 35%(7/20)bdghij
64 70%(14/20)bde 60%(12/20)bde 45%(9/20)bedf 20%(4/20)b

 

Means for germination or conversion followed by one or more of the same letters
were not significantly difi‘erent based on a chi-square at p50.05.

Table 3-4: Comparison of germination rates in soil of encapsulated somatic embryos with
eitherMSmediumothOmdefi‘ectsonthegaminafionratesafierstomgeat4‘C.

 

 

 

 

ays of storage Germination response
at 4 °C alg’nate encapsulation with
MS medium H30

0 53% (16/30) NS 53% (16/30) NS

9 40% (8/20) NS 45% (9/20) NS
21 45% (9/20) NS 50%(10/20) NS
30 50% (5/ 10) NS 40% (4/10) NS
41 50% (5/10) NS 40% (4/10) NS
50 40% (4/10) NS 40% (4/10) NS
64 38% (3/8) NS 25% Q8) NS

 

 

 

 

 

Nsmdicatedthatthegaminafionratesofembryosencapadatedwhhalginatein
eitherMSmediumoerowerenotsigmfieamlydifl‘erentbasedonachi-squm

at p$0.0S.

 

Figure 3-2: Somatic embryogenesis alter suspension plate-out on MS medium with
0 3 mg/LNAA and 0.1mg/L ABA after 31 days.

 

 

Figure 3-4: Somatic embryo (4 mm) directly developed into complete plantlet on the
suspension plate-out medium.

Figure 3-5: Somatic embryo with bipolar converted into cotyle ons an roots srrn -
taneously, 6 days after being transferred to homrone-free MS medium
(length: 14 mm).

..5.‘

Figure 3-6: Somatic embryos develop into complete plantlet, 9 days after being transferred
to hormone-free MS medium (length: 19 mm).

 

116

 

Figure 3-7: Secondary embryogenesis were observed from the primary somatic embryo
(8 mm), 14 days after being transferred to hormone-lice MS medium.

I r—

 

.- .
Figure 3-8: Isolated somatic embryos (length: 0.5-4 mm).

 

4-5 mm.

0'19‘3‘1rr) n,‘
‘ f (£170 :1..-

.- fw‘f“

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—
—
— .
—
r
——

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l7
llll

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lllllll‘

9
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Figure 3-10: The diameter of each gel bead rs approximately 4-5 mm.

Figure 3-12: An encapsulated embryo that germinated afier the seventh day on agar plate,
with betalain pigment on the hypocotyls.

 

 

Figure 3-13: An encapsulated embryo that converted into a plantlet afier fourteenth day
on hormone-free MS agar plate, with betalain pigment on the hypocotyl
(length: 15 mm).

 

simultaneously after 21 days of storage at 4 °C (length. 14 mm).

120

#1me mum

Figure 3-15: An encapsulated embryo that germinated out (with radicle elongation) of
encapsulated coat afier 64 days of cold storage without transfer. Capsule
is about 4 mm in diameter.

 

CHAPTER 4
EVALUATION OF ALTERNATIVE NITROGEN SOURECES FOR
SUGARBEET SUSPENSION CULTURE PLATINGS IN
DEVELOPNIENT OF CELL SELECTION SCHEMES
ABSTRACT
Nitrogenous impurities are negative quality factors in sugarbeet (Beta vulgaris L.)
processing. In order to identify nutrient media for cell selection of biochemical mutants or
transgenics that might have reduced levels of these impurities, the ability of eight
endogenous compounds to serve as sole nitrogen source in suspension culture platings

was evaluated with clone REL-1. Nitrate (N03), ammonium (NI-L), glutamine (GLN),
glutamate (GLU), and urea as sole nitrogen sources were moderately supportive of plating
callus growth based on fi'esh weight compared with the standard 60 mM mix of N03 and
NIL in Murashige-Skoog (MS) medium Proline (PRO) was poorly supportive, and
glycine betaine (BET) and choline (CHO) were non-supportive. No callus growth
occurred when GLN, GLU or BET was provided as the sole carbon or carbon plus
nitrogen source. Platings on either N02 or NH. as sole nitrogen source did not difi'er in
sensitivity to the nitrate uptake inhibitor phenylglyoxal (PGO), suggesting that PGO lacks
the specificity for use in selection for mutants of nitrate uptake. The ability of GLN or
GLU to be used as the nitrogen source may preclude their use for selection of genetic
variants accumulating less of these processing impurities. However, mutants or transgenics

able to utilize GLN, GLU or BET might be selectable on media containing any one of

121

1 22
these as carbon, nitrogen or carbon plus nitrogen source, respectively, that is incapable of

supporting wild type cell growth.

INTRODUCTION

Somatic cell selection has potential for identifying novel single gene variations in crop
species (Duncan and Widholm, 1986). The extent of the correlation between in vitro and
whole plant responses, as well as the recognition of cell selective regimes targeting
specific metabolic and regulatory areas, may limit improvement of the range of crop
characteristics by the method.

The recovery of monogenic dominant sulfonylurea herbicide resistant plants by using
somatic cell selection has been demonstrated in sugarbeet (Saunders et al., 1992). Their
system involved a diploid clone (REL-1) bred for fine suspension culture texture and easy
regeneration of nonvitreous shoots, as well as an acute (one time) exposure of plated
suspension cell clusters to a lethal concentration of chlorsulfuron, a specific inhibitor of
acetolactate synthase. This selection step was performed on a shoot regeneration medium
to maximize chances of recovering shoots from any surviving colonies.

In order to increase the eficiency of industrial sucrose extraction, reducing the
concentrationofimpuritiesintheharvested sugarbeetroothasbeenabreedingobjective
(Smith, 1988; Smith and Martin, 1989). These impurities are the small molecules that are
not removed by precipitation at the temperature and pH conditions employed in the
sucrose purification process. The major nitrogenous impruities or their derivatives in
sugarbeet molaSses are GLN, GLU, and BET. One way to reduce the level of processing

impurities would be to detemrine if the individual impurities would serve as sole nitrogen

1 23
sources in vitro. If any major nitrogen impurity would not serve as a sole nitrogen source,
then a medium containing such an impurity could be used as a positive selective regime for
cell variants that had acquired the ability to utilize that compound. A precedent for this
approach in higher plants was the selection of a glycerol utilizing mutant in tobacco cells
that was later recovered in sexual progeny (Chalefl‘ and Parsons, 1981).

In contrast, if individual impurities or other endogenous metabolites could serve as sole
nitrogen sources for wild-type cultured cells, then such media could be used in forrmrlation
of a difi‘erent kind of positive cell selection scheme that relies on a specific inhibitor of an
immediate assimilation step of that sole nitrogen source. Use of the single source of
nitrogen would be important to make the cells susceptible to such an inhibitor. Expression
of a mutation or epigenetic change conferring resistance to the inhibitor could permit cell
survival. Heimer and Filner (1970) recovered a tobacco cell line resistant to growth
inhibition by threonine when nitrate was the sole nitrogen source. Sugarbeet mutants with
resistance mechanisms involving enzyme overproduction might have a reduced
concentration of the respective impurity at the whole plant level. Furthermore, use of sole
nitrogen sources could help characterize mutants in nitrogen metabolism, such as in
soybean where difl‘erent classes of urease mutants are defined according to tissue
specificity (Polacco et al., 1989).

Thus, in order to determine their potential role in somatic cell selection schemes,
glutamine, glutamate, and glycine betaine were evaluated for use as either a sole nitrogen,
carbon, or a combination carbon and nitrogen source. Several other endogenous nitrogen
compounds were evaluated as sole nitrogen sources because of interest in devising

selection schemes using inhibitors for recovery of overproducing mutants for steps in

124
nitrogen assimilation, with the goal of ultimately improving nitrogen use eficiency.
Lastly, phenylglyoxal (PGO), an inhibitor of nitrate assimilation (Dhugga et al., 1988),
was tested with either nitrate or ammonium as sole nitrogen source to evaluate its

suitability as a selective agent.

MATERIALS AND METHODS

All experiments were performed with suspension cultures of the diploid sugarbeet clone
REL-1. Suspension cultures were initiated fiom leaf disc callus (Saunders et al., 1992).
All experimental media contained Murashige and Skoog (MS) (1962) inorganics, 3 %
sucrose, 100 mg/L myo-inositol, 10 mg/L thiamine‘HCl, 1.0 mg/L nicotinic acid, 1.0 mg/L
pyridoxine'HCl. 0.25 mg/L 6-benzyladenine (BA), and 0.9 % Difco Bacto agar (w/w).
Nitrate (N 03), ammonium (NI-14), glutamine (GLN), glutamate (GLU), proline (PRO),
urea, glycine betaine (BET), and choline (CHO) were tested as sole nitrogen sources at
15, 30, 60, and/or 90 mM KN03 (20 mM) and NH4N03 (40 mM) togetherwere used as
the standard MS nitrogen source, except when alternative sole nitrogen sources were
tested. In such cases the sources of potassium were from the 2.0 mM KI-12P04 in MS, any
KOH used in pH adjustment, or stoichiometric quantities from KNO; used in providing
the various levels of N03. NIL was provided as the chloride salt and was accompanied by
halfthat concentration of succinic acid. Sucrose was omitted in tests of alternative carbon
(C) or carbon & nitrogen (C+N) sources. GLN, GLU or BET were included at 33 and
66mMand comparedwith29mMsucrosewhenthesetbreecompoundsweretestedasC

or C+N sources. GLU was provided as the sodium salt, BET, CHO and PRO as the free

bases. Individual K“, Na+ or Cl' concentrations were not adjusted to maintain equivalency.

1 25

Suspension cultured cells used as inoculum were washed once through decanting in
medium lacking either N, C, or C+N sources, then passed through a stainless steel sieve
with 830 uM openings using additional wash medium Solutions of all N, C, or C+N
sources and of PGO (Sigma Chemical Co., St. Louis, M0) were adjusted to pH 5.95 with
KOH or HCl prior to filter sterilization, and added during cooling of the medium, which
had undergone pH adjustment with KOH before autoclaving (15 psi, 121°C, 20 min).

Thirty-five mL of medium was dispensed into 100 x 20 mm Falcon disposable plastic
Petri plates. Plates were inoculated with 1.0 mL of log phase suspension using disposable
plastic Pasteur pipettes. Each plate received, fiom experiment to experiment, about one
hundred to three hundred viable cell clusters (20-35 mg total dry weight). Afler
inoculation, plates were sealed with two Parafilm strips and incubated in dim light (less
than 5 umol rn’2 s"1 from fluorescent lamps) at 25°C. Final fi'esh weight was measured and
each treatment was replicated 8 to 13 times, and experiments were performed with similar
results 2 or 3 times each. Final callus fresh weight for each medium treatment were
subjected to AN OVA for a randomized complete block design and the LSD (P=0.05) was

used to compare individual treatments.

RESULTS
The N sources N03, NI-Ir, GLN, GLU, and urea supported callus growth, but not as
well as the standard 60 mM Nos/NIL nitrogen mixture in MS medium (Figure 4-1).
Callus growth on BET, CHO, and PRO was not significantly difl‘erent (P=0.05) fiom the

nitrogen-flee control (Figure 4-1).

1 26
The nitrogenous processing impurities GLN, GLU, and BET evaluated for use as sole
C or C+N sources did not support growth (Figure 4-2).
Since either N03 or NI-L as sole nitrogen source supported moderate growth, an
experiment was conducted to test the specificity of inhibition of callus growth by PGO
with each nitrogen source, alone and in combination. PGO at 2100 uM almost completely

inhibited growth regardless of source of N. PGO at 700 uM also inhibited, but inhibition

was greater with N0; than with NIL (Figure 4-3).

DISCUSSION

In order to identify opportunities for selecting biochemical mutants at the cell level,
alternative nitrogen sources were investigated for plated callus growth of sugarbeet
suspensions. Mutants of interest for use in sugarbeet improvement include those for
modified (1) metabolism of processing impurities that might lead to reduced levels of
those impurities at the plant level in harvested tap roots, and (2) nitrogen assimilation,
given the recognized major influence of nitrogen on sugarbeet root yield, sucrose
percentage, and processing eficiency.

A wide range (15, 30, 60, and/or 90 mM) of endogenous nitrogen compounds was
tested as sole nitrogen sources. These included processing impurities (GLN, GLU, BET),
a metabolite (PRO, fiom GLU), a precursor (CHO, to BET), inorganic forms (N03, NH4)
and an intermediate sometimes used in crop fertilization (urea). All of the tested
compounds except BET, CHO and PRO were at least moderately supportive of growth in
the presume of the MS mixture of N03 and NH4. Sugarbeet callus could utilize N03 as

sole nitrogen source, unlike sugarcane callus which could not rely on it (V eith and Komor,

1 27
1991). Sugarbeet was not capable of utilizing BET for the growth of callus from plating
This may be related to the apparent lack of BET metabolism in the whole plant (Hanson
and Wyse, 1982).

When processing impurities GLN (largely converted to GLU and pyroglutamate in the
heat of the extraction and purification process), GLU and BET were also evaluated as sole
C and C+N sources for platings, none was efi‘ective. Hence, these could be used for
positive selection to recover mutants that have acquired the ability to utilize these
impurities. Such mutants might also possess altered metabolism and altered accumulation
of the impurities at the whole plant level. Media with GLN or GLU as C or C+N source,
or BET as N, C or C+N source might also be used to select for transgenic cells carrying
genes permitting utilization of any of these processing impurities. For example, genes for
catabolism of BET in Rhizobiwn melliloti (Le Rudulier et al., 1984) could be cloned and
potentially used in transformation of sugarbeet.

An inhibitor of a specific step of nitrogen assimilation, in combination with a sole
nitrogen source just upstream of the step sensitive to the inhibitor, should permit isolation
of resistant mutants that produce excess enzyme or an altered enzyme insensitive to the
inhibitor. Mutants overproducing the target enzyme by gene amplification or upregulation
could be of greater value for crop improvement than amino acid sequence changes
resulting in reduced inhibitor binding, provided agronomic performance or crop quality
were improved in the former. Additionally, resistance to the inhibitor might be used as a
pleiotropic marker for easy and early identification of the overproducing segregates in
progeny. The possibility of such an indirect selection scheme, where selection for

resistance to an inhibitor could lead to isolation of a mutant for altered nitrogen

 

1 28

assimilation, was investigated in this research. Dhugga et al. (1988) demonstrated that
N03 and NFL as sole N sources, the specificity of growth inhibition by PGO, inhibited
N03 uptake. Unfortunately, the inhibition of grth by PGO was not found to be specific
to NO; as a nitrogen source (Figure 4-3), and thus selection for resistance to PGO may
not be useful for recovering nitrate uptake mutants. A further possibility for this kind of
indirect selection for enzyme overproducing mutants of nitrogen assimilation (and possibly
of agronomic performance) would involve concomitant use of urease inhibitor N-(n-butyl)
thiophosphoric triamide (Bremner and Krogrneier, 1988) with urea as sole N source.

Selection schemes to recover biochemical mutants with new characteristics or higher
levels of endogenous enzyme activity are of interest because their occrurence in crop or
higher plants is limited, where descriptions of hypomorphs or millimorphs predominate.
Reports on in vitro recovery of mutants in nitrogen metabolism that involve enzyme
overproduction, acquisition of function, or beneficial efi'ects on agronomic performance
are even less common. Sabir et al. (1992) found that an apparent overproducer of
glutamate dehydrogenase was recovered as a somaclonal variant in sugarbeet. Donn et al.
(1984) recovered a cell line of alfalfa overproducing glutamine synthetase (GS) following
somatic cell selection for resistance to the GS inhibitor phosphinothricin The basis for
resistance was amplification of the GS gene. The trait was interpreted to be dominant as it
was expressed in hybrid protoplasts (Deak et al., 1988).

Some of the media with alternative sole N sources were completely or partially
inefi‘ective in supporting callus growth, with or without use of inhibitors, for example,
PRO, BET and CHO. Such media could be used to isolate transgenic cells expressing

heterologous genes for utilization of endogenous nitrogenous compounds, and/or to select

1 29
transgenic cells carrying “activation tagging” constructs generating dominant phenotypes

by the overexpression of genes (Hayashi et al., 1992).

130

REFERENCES

Bremner, J .M. and MI. Krogrneier. 1988. Elimination of the adverse efi‘ects of urea
fertilizer on seed germination, seedling growth and early plant growth in soil. Proc.
Natl. Acad. Sci. 85: 4601-4604.

Chalet}; RS. and MF. Parsons. 1978. Isolation of a glycerol-utilizing mutant of Nicoticma
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Dealg M., G. Donn, A Feher, and D. Dudits. 1988. Dominant expression of a gene ampli-
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Dhugga, KS., J.G. Waines, and RT. Leonard. 1988. Nitrate absorption by com roots.
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Donn, G., E. Tischer, J.A Smith, and HM. Goodman. 1984. Herbicide-resistant alfalfa
cells: an example of gene amplification in plants. J. Mol. Appl. Genet. 2: 621-635.

Duncan, DR and J .M. Widholm 1986. Cell selection for crop improvement. Plant
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Hanson, AD. and R Wyse. 1982. Biosynthesis, translocation, and accumulation of
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Hayashi, H., I. Czaja, H. Lubenow, J. Schell, and R Walden 1992. Activation of a
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-1353.

Heimer, Y.M. and P. Filner. 1970. Regulation of the nitrate assimilation pathway of
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152-165.

Le Rudulier, D., AR Strom, AM. Dandekar, L.T. Smith, and RC. Valentine. 1984.
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Murashige, T. and FA Skoog. 1962. A revised medium for rapid growth and bioassay
with tobacco tissue cultures. Physiol. Plant. 15: 476-497.

Polacco, J.C., AK. Judd, J.K Dybing, and SR Cianzio. 1989. A new mutant class of
soybean lacks urease in leaves but not in leaf-derived callus or in roots. Mol. Gen.
Genet. 217: 257-262.

131

Sabir, A, H.G. Newbury, G. Todd, J. Catty, and B.V. Ford-Lloyd. 1992. Determination
of genetic stability using isozymes and RFLPs in beet plants regenerated in vitro. Theor.
Appl. Genet. 84: 113-117.

Saunders, J .W., G. Acquaah, K.A Renner, and W.P. Doley. 1992. Monogenic dominant
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1360.

132

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CHAPTER 5

EVALUATION OF SOLE NITROGEN SOURCES FOR SHOOT CULTURES
AND LEAF DISC CULTURES OF SUGARBEET

ABSTRACT

Eight endogenous nitrogen-containing sources (nitrate, ammonium, glutamine,
glutamate, urea, proline, glycine betaine, and choline) were examined for their ability to
serve as sole nitrogen source for shoot culture as well as leaf disc culture of sugarbeet
(Beta vulgaris L.) clone REL-1. Nitrate (N03), ammonium (NH4), urea, and glutamine
(GLN) as sole nitrogen sources were moderately supportive of shoot multiplication and
fresh weight increase of shoot culture growth compared to the -Murashige-Skoog (MS)
nitrogen mix of 40 mM N03 and 20 mM NH4. Glutamate (GLU) and proline (PRO) were
at best poorly supportive, and glycine betaine (BET) and choline (CHO) were non-
supportive, of shoot culture growth Callus initiation from leaf discs was moderately
supported only by sole nitrogen sources N03, NH4, urea, GLN, and PRO. Shoot
regeneration from the disc callus was further supported on media with GLN, N03, urea or
PRO, although at least three times more shoots per callusing disc were produced on the
MS nitrogen mix Overall, the MS nitrogen mix was superior or equal to any ofthe single
nitrogen sources. However, single nitrogen source media with N03, NH4, urea, GLN or
PRO could be used for shoot cultures of mutants with impaired nitrogen assimilation or in

comparative physiology studies.

135

 

 

136

INTRODUCTION

Commonly used basic media for plant tissue culture use various sources and quantities
of nitrogen (Murashige and Skoog, 1962; Blaydes, 1966; Gamborg et al., 1968; Schenk
and Hildebrandt, 1972; Lloyd and McCown, 1980). Most basic media have a mixture of
nitrate and ammonium, and some have an additional form of organic nitrogen Comparison
of growth or developmental responses on these media permits few conclusions about
quantitative or qualitative differences in nitrogen content because the efi‘ects of many other
nutritional factors confound interpretations.

Nitrogen is a very important nutrient for sugarbeet crop yield and processing quality.
Glutamine, glutamate and glycine betaine are nitrogenous impurities in the harvested beet
that interfere with industrial sucrose extraction eficiency (Smith, 1988). Glutamine and
glutamate, but not glycine betaine, were found to support sugarbeet plating callus growth
in a test of eight sole nitrogen sources conducted in support of efi‘orts to develop cell
selection regimes for mutants in nitrogen assimilation (Chapter 4). In order to determine
the feasibility of using shoot and leaf disc systems to study growth and metabolism in wild-
type and prospective nitrogen metabolism mutants, we investigated the responses of

cmltured sugarbeet shoots and leaf discs.

137

MATERIALS AND METHODS

Plant materials: All experiments were performed with shoot cultures and leaf discs of
sugarbeet clone REL-1, a diploid self-fertile annual clone, which is a superior regenerator
of shoots from leaf callus, and easily maintained by in vitro shoot culture (Saunders et al.,
1990).

Media: All media were formulated according to Murashige and Skoog (MS) (1962)
inorganics, 10 mg/L thiamine‘HCl, 1.0 mg/L nicotinic acid, 1.0 mg/L pyridoxine‘HCl, 100
mg/L myo-inositol, 3 % sucrose, and 0.9 % agar w/v. Concentration of 6-benzyladenine
was 1.0 mg/L for leaf disc culture; 0.25 mg/L for shoot culture. Nitrate (N03), ammonium
(NT-1.), glutamine (GLN), glutamate (GLU), proline (PRO), urea, glycine betaine (BET),
and choline (CHO) were tested as sole nitrogen sources at 15, 30, 60, and/or 90 mM in
the cases of both shoot cultures and leaf disc cultures. N03 (40 mM) and NH4 (20 mM)
together were used as the standard MS nitrogen, except when alternative sole nitrogen
sources were tested. A nitrogen-flee medium served as a negative control. In such cases
the sources of potassium were fi'om the 2.0 mM KH2P04 in MS, any KOH used in pH
adjustment, or stoichiometric quantities fipm KNO; used in providing the various levels of
N03. NI-L was provided as the chloride salt and was accompanied by half that
concentration of succinic acid. GLU was provided as the sodium salt, BET, CHO and
PRO as the fiee bases. Individual K, Na“ or Cl‘ concentrations were not adjusted to
maintain equivalency.

Shmt culture: Cultures for shoot multiplication had been established fi’om axillary shoots

on a flower stalk in 1985 and subcultured by division every 2-3 months. Each 20- by 100-

 

1 38

mm Petri dish contained three shoots (initial mean fresh weight of a shoot was
approximately 0.15 g) on 35 m1 of MS medium with BA 0.25 mg/L, and were grown at 25
°C and under 10 to 20 umol m'zs'l fiom fluorescent lamps. For the media comparison, total
fresh weight of shoots per plate was measured alter 35 days and the threesomes were
subcultured to fresh respective media. After a further 26 days, each plate’s shoots were
measured to produce a 61 day cumulative fi'esh weight as well as shoot mrmber increase.
Theminimumsizeforashootwa505 cm. Fmalmediumprasalsomeasuredonthe
61" day.

Leaf disc culture: Leaf discs (8 mm diameter) were taken fi'om incompletely expanded
leaves of greenhouse-grown vegetative plants and disinfected with a dilute (1 :5 v/v)
commercial (5.25 % w/v) hypochlorite (NaOCl) bleach containing 100 mg/L sodium
laurylsulfate.One8mmleafdiscwasplacedon35mlofMSagarmediumwith1mg/L
BA in a 20- by 100- mm Falcon disposable Petri dish Plates were kept at 30 °C in the
dark (Saunders et al., 1992). Percent of discs callusing was observed after 42, 61, and 86
days of incubation. Mean number of shoots per callusing disc was counted after 106 days
of culture. Expansion of leaf discs was measured afier 15 days, and expressed as increase

in diameter.

Each experiment with 10 replications was performed 3 times with similar results. Final
fresh weight, shoot multiplication factor, percentage of discs callusing, mean shoots per
callusing disc, and expansion of leaf discs for each medium treatment were subjected to

AN OVA and the LSD (P=0.05) was performed to compare individual treatments.

 

 

139

RESULTS AND DISCUSSION
Shoot culture: Shoot culture response to the eight sole nitrogen sources as measured by
cumulative fi'esh weight of initial culture (35 days) and subculture (26 days) (Figure 5-1),
and shoot number increase (Figure 5-2) indicated that N03, NI-L, GLN and urea
supported growth at best 81 % as well as the MS nitrogen mix GLU, PRO, BET and
CHO were inefi‘ective. In general, the intensity of shoot multiplication on each medium
corresponded to the respective fresh weight response.

The final medium pH in the shoot cultures (Figure 5-3) was not associated with the
relative increase of shoots and fresh weight. The final pH values with N0; and GLU were
above 6; MS, nitrogen-fies (MS-N), N114 and urea were around -5; GLN, PRO, BET and
CHO were below 5. Gamborg and Shyluk (1970) reported that there was no apparent
correlation between final pH and growth of the soybean cells with the various ammonium
salts, when the soybean cells grew on ammonium salts as the sole nitrogen source.
Therefore, the abilities of sole nitrogen sources to support growth do not seem to be
dependent on the value of the final medium pH.

Leaf disc culture: Callus was initiated from leaf disc only after at least 25 days of
inoculation. Callus initiation response was not designed to involve amount of callus
produced. GLU, BET, CHO and nitrogen-free media initiated no callus within 86 days of
observation (Figure 5-4). All leaf discs had initiated callus from NFL (30 mM), GLN (15
mM) and MS media after an inoculation period of 42 days (Figure 5-4). Overall, callus

initiation slowed after six weeks. Furthermore, shoots regenerated from callusing leaf

 

1 40

discs on MS, N03 (30, 60 mM), urea (15, 60 mM), GLN (15, 30, 60 mM) and PRO (30,
60 mM) (Figure 5-5) within 106 days of culture. GLN was the best sole nitrogen source
for shoot production, but it was only about a third as productive as the MS nitrogen mix
Shoot production from callusing discs was lower at the higher GLN concentrations. All
media that induced callus from leaf discs also induced some shoot regeneration fi'om that
callus, except for 30 mM urea and the NIL media (Figure 5-6, Table 5-1). The
nonuniform number of discs that callused, and the low mrmber of callusing discs that
regenerated shoots on some media made statistical analysis of shoot regeneration
impractical. Although the media with urea and NIL induced high percentages of disc
callusing, the frequency of regenerated shoots was low and zero, respectively. Numerous
studies have shown that most plants respond more favorably to nitrate than to ammonium
as the sole nitrogen source. Filner (1966) found that ammonium did not support growth
in cultured tobacco cells. Yatazawa and Furuhoshi (1968) reported that rice callus grew
on agar media containing ammonium citrate but not on ammonium sulfate. Anyway, it
really depends how the NIL is presented to the cultures, i.e., the companion anion. Some
studies apparently not aware of this.

Leafdisc expansion (Figure 5-7) was not directly associated with callus initiation; note
especially the response to N03 vs. PRO (Figure 5-4). All leaf discs attained their final
expansion during the initial 15 days of inoculation. However, the results indicated that the
N03, NIL and GLN sole nitrogen media were similar to the MS nitrogen mix medium for
leaf disc expansion.

Unlike the shoot culture tests, PRO responded positively in the callus initiation of leaf

disc. Moreover, shoot regeneration response on leaf disc callus did not correspond to

141

shoot culture response; only GLN and N03 induced a moderate response relative to the
100 % obtained with the standard MS nitrogen mix.

Hanson and Wyse (1982) reported that BET could not be utilized for cell culture
growth, it might be associated with the apparent lack of BET utilization in the whole
plant. Thus, ability of any sole nitrogen source to support growth in leaf disc and/or shoot
culture indicates that the nitrogen source is being metabolized. This might also reflect in
difl‘erential nitrogen response at the whole plant level. From this research, callus and
shoots could be regenerated using GLN as a sole nitrogen source to produce
independently-derived shoot regenerates. This could permit a degree of quantitative cell
selection for ability to assimilate GLN, which might be reflected later in altered nitrogen

response at the whole plant level.

142

REFERENCES

Atanassov, AI. 1980. Method for continuous bud formation in tissue culture
of sugar beet (Beta vulgaris L.). Z. Pflanzenzucht. 84: 23-29.

Blaydes, DR 1966. Interaction of kinetin and various inhibitors in the growth
of soybean tissue. Physiol. Plant. 19: 748-753.

Coumans-Gilles, M.P., C. Kevers, M. Coumans, E. Ceulemans, T.H. Gaspar.
1981. Vegetative multiplication of sugarbeet through in vitro culture of
inflorescence pieces. Plant Cell Tissue Org. Cult. 1: 93-101.

F ilner, P. 1966. Regulation of nitrate reductase in cultured tobacco cells.
Biochirn. Biophys. Acta 1 18: 299-310.

Freytag, AH., 80 Anand, AP. Rao-Arelli, and L.D. Owens. 1988. An
improved medium for adventitious shoot fomration and callus induction in
Beta vulgaris L. in vitro. Plant Cell Rep. 7: 30-34.

Gamborg, O.L., RA Miller and K. Ojima. 1968. Nutrient requirements of
suspension cultures of soybean root cells. Exp. Cell Res. 50: 151-158.

Gamborg, 0L. and JP. Shyluk. 1970. The culture of plant cells with ammo-
nium salts as the sole nitrogen source. Plant Physiol. 45: 598-600.

Hanson, AD. and R Wyse. 1982. Biosynthesis, translocation, and accumu-
lation of betaine in sugarbeet and its progenitors in relation to salinity.
Plant Physiol. 70: 1191-1198.

Hooker, M.P. and M.W. Nabors. 1977. Callus initiation, growth, and organo-
genesis in sugarbeet. Z. Pflanzenphysiol. 84: 237-246.

Lloyd, G. and B. McCown. 1980. Commercially feasible micropropagation
of mountain laurel, Kalmr'a Ianfolia, by use of shoot tip culture. Intl. Plant
Prop. Soc. Proc. 30: 421-427.

Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and
bioassays with tobacco tissue culture. Physiol. Plant. 15: 473-497.

Owens, L.D. and DR Eberts. 1992. Sugarbeet leaf disc culture: an improved
procedure for inducing morphogenesis. Plant Cell Tissue Organ Cult. 31:
195-201.

143

Saunders, J.W., G. Acquaah, K.A Renner and W.P. Doley. 1992. Mono-
genic dominant sulfonylurea resistance in sugarbeet from somatic cell
selection. Crop Sci. 32: 1357-1360.

Saunders J .W. and W.P. Doley. 1986. One step shoot regeneration from
callus of whole plant leaf explants of sugarbeet lines and a somaclonal
variant for in vitro behavior. J. Plant Physiol. 124: 473-479.

Saunders, J.W., W.P. Doley, J.C. Theurer, et al. 1990. Somaclonal variation
in sugarbeet. In Y.P.S., Bajaj. (ed). Biotechnology in agriculture and forestry.
Vol. II. Somaclonal variation in crop improvement I. Springer-Veriag, Berlin
Heidelberg. pp.465-490.

Saunders, J .W. and K. Shin. 1986. Germplasm and physiologic efi‘ects on
induction of high-frequency hormone autonomous callus and subsequent
shoot regeneration in sugarbeet. Crop Sci. 26: 1240-1245.

Saunders, J .W., C]. Tsai and E. Samper. Evaluation of alternative nitrogen
and carbon sources for sugarbeet suspension culture platings in development
of cell selection schemes. In Vitro Cell. Dev. Biol. Submitted.

Schenk, RV. and AC. Hildebrandt. 1972. Medium and techniques for induc-
tion and growth of monocotyledonous and dicotyledonous plant cell cul-
tures. Can. J. Bot. 50: 199-204.

Smith, GA 1988. Efi‘ects of plant breeding on sugarbeet composition In
Clarke, M.A and M.A Godshall (ed) Chemistry and processing of sugar-
beet and sugarcane. Elsevier, Amsterdam. pp 9-19.

Yatazawa, M., and K. Furuhoshi. 1968. Nitrogen sources for the growth of rice
callus tissue. Soil Sci. Plant Nutr. 14: 73-79.

144

 

 

 

 

 

 

 

 

 

 

 

 

 

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