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This is to certify that the

dissertation entitled

Development of Fusarium Resistance of Asparagus
via Tissue Culture And Gene Transformation

presented by

Yinghui Dan

has been accepted towards fulﬁllment
of the requirements for

Doctor of Philosophy . Botany and Plant Pathology
degree in

 

 

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DEVELOPMENT OF F USARIUM RESISTANCE OF ASPARAGUS

VIA TISSUE CULTURE AND GENE TRANSFORMATION

By

Yinghui Dan

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1994

ABSTRACT

DEVELOPMENT OF F USARIUM RESISTANCE OF ASPARAGUS
VIA TISSUE CULTURE AND GENE TRANSFORMATION

By

Yinghui Dan

Asparagus oﬁ'icinalis L. is an economically important vegetable crop that is produced -
worldwide. However, a disease syndrome known as asparagus decline, primarily caused
by Fusarium oxysponun and F. proliferatum, decreases the annual yields of asparagus
over time, and is found throughout the asparagus growing regions of the world. The use
of conventional methods including chemical and cultural methods, and traditional
breeding of resistance in the host for controlling Fusarium spp. has been limited. The
objective of this study was to explore the development of Fusarium resistance of
asparagus via tissue culture and gene transformation, and determine the genetic base of
the resistance. A procedure of isolation, culture and plant regeneration of callus-derived
protoplasts of Asparagus oﬁicinalis cv. Lucullus 234 was developed. Protocols were also
developed for vegetative micropropagation and somaclone production of ’Lucullus 234’.
One hundred and twenty somaclones screened for resistance to F. oxysporum and F.
proliferatum in the greenhouse had signiﬁcantly increased levels of resistance over the
vegetatively micropropagated ’Lucullus 234’ parental plants. Two somaclonal lines

demonstrating increased resistance from the ﬁrst cycle of screening, R7 and R4, were

rated as highly resistant compared with the parental plants when rescreened with F.
oxysporum in the greenhouse after they were vegetatively micropropagated. Cytogenetic
studies showed that R7 and R4 were diploid with normal chromosomal numbers (2n =20)
as ’lucullus 234’ parental cultivar (2n=20). The genomic ﬁngerprints obtained by
randomly ampliﬁed polymorphic DNAs (RAPDs) technology indicated that resistant
somaclonal lines, R7 and R4, may have carried DNA sequence mutations, as their DNA
sequences varied from the ’Lucullus 234’ parental plants. Spear segments of ’Lucullus
234’ were transformed using the binary vector Agrobacterium tumefaciens
LBA4404/pB1121 containing an anti-bacterial and -fungal gene (Shiva-1) encoding a lytic
peptide. A single transgenic plant (TI 1) was obtained. This plant displayed GUS
activity, and Southern blot suggested the presence of two copies of the Shiva-1 gene
integrated into the genome of T11. An in-vitro antibacterial assay showed that T11
signiﬁcantly inhibited the growth of E. coli, indicating possible production of Shiva-1
peptide in T11. The vegetatively micropropagated plants of T11 showed a signiﬁcant
increase in resistance to F. oxysporum in the greenhouse in comparison with vegetatively

micropropagated nontransgenic plants.

iv

To

My parents, whose unselﬁsh love supported me through my education.

My husband, for his endless love, understanding and support.

My son, for his love, warm smiles and innocent ways.

ACKNOWLEDGMENTS

First and foremost, I would like to thank my parents, Zhongli Dan and Huanwen
Chen, and my husband and son, Liang and Daniel, for all of their love and support over
the years. Without their emotional support the completion of this degree would not been
possible.

Secondly, I would like to thank my major professor, Dr. C. Stephens, for her
support, guidance and patience, and for giving me the freedom to explore on my own
through the years.

I would like to thank my committee members, Drs. D. Fullbright, 1. Hancock, C.
Stephens and R. Allison, for their useful comments and help.

Finally, thank you to all of you within the department who I have had the pleasure

to learn with and from, both in and out of the laboratory.

TABLE OF CONTENTS
Page
LIST OF TABLES ....................................... ix
LIST OF FIGURES ....................................... xi
LIST OF ABBREVIATIONS ................................ xiii
INTRODUCTION ........................................ 1
LITERATURE CITED ..................................... 9

CHAPTER 1: Optimal Conditions for Growth and Plant Regeneration of Asparagus
(Asparagus Qﬁ‘icinalis L. cv. Lucullus 234) from Callus-Derived

Protoplasts.
ABSTRACT .......................................... 17
INTRODUCTION ....................................... 18
MATERIALS AND METHODS .............................. 20
Plant material ........................................ 2O
Isolation of protoplasts .................................. 20
Culture of protoplasts ................................... 21
Plant regeneration of calli from single protoplasts ................. 24
RESULTS AND DISCUSSION .............................. 25

Effects of different media on plating efﬁciency and
colony formation in liquid and agarose layer culture ................ 25

Effects of osmotic condition of liquid media on plating
efﬁciency and colony formation in bead culture ................... 27

vii

Effects of hormone concentrations of liquid media on

plating efﬁciency and colony formation in bead culture .............. 32
Effects of types of culture on plating efﬁciency and colony formation ...... 32
Comparison of different media on shoot induction .................. 35
CONCLUSION ........................................ 36
LITERATURE CITED .................................... 40

CHAPTER II: The Development of Asparagus oﬂicinalis cv. Lucullus 234
Somaclones with High Levels of Resistance to Fusarium spp.

ABSTRACT .......................................... 43
INTRODUCTION ....................................... 44
MATERIALS AND METHODS .............................. 50
Production of somaclones ................................ 50
Vegetative micropropagation of ’Lucullus 234’ ................... 51

The ﬁrst cycle of screening for higher levels of
Fusarium resistance in somaclones in the greenhouse ................ 52

Test stability of the Fusarium resistance in the more

resistant somaclones selected from the ﬁrst cycle .................. 5 3
Cytogenetic study of the resistant somaclones .................... 54
Determining the genetic bases of the resistant
somaclones by the RAPDs ................................ 54
RESULTS AND DISCUSSION .............................. 56
Production of somaclones ................................ 5 6
Vegetative micropropagation of ’Lucullus 234’ ................... 57
Screening of somaclones for Fusarium resistance .................. 61

Measuring genetic variability in the resistant somaclones ............. 68

viii

LITERATURE CITED .................................... 78

APPENDIX: The Transformation of Asparagus with A Synthetic
Gene Encoding A Novel Lytic Peptide

ABSTRACT .......................................... 83
INTRODUCTION ....................................... 84
MATERIALS AND METHODS .............................. 88

Plant transformation with Agrobacterium ....................... 88

Selection of transfonnants using Flourimetric GUS assay and NPTII Elisa . . . 9O

Conﬁrmation of transformed plants by Southern Blot ................ 91
In-vitro anti-bacterial activity assay of transgenic plant T11 ............ 93
Evaluation of the vegetatively micropropagated plants of
T11 in response to F. oxyspomm in the greenhouse ................. 94
RESULTS AND DISCUSSION .............................. 95
Regeneration and selection of transgenic plants ................... 95
Conﬁrmation of transgenic plants ............................ 97
Antibacterial activity of transgenic plant T11 ..................... 100

Evaluation of the vegetatively micropropagated plants
of T11 in response to F. oxyspomm in the greenhouse ............... 102

LITERATURE CITED .................................... 105

ix

LIST OF TABLE

TABLE PAGE

1.1 Effects of different media and types of culture on plating
efﬁciency and colony formation of asparagus protoplasts .......... 26

1.2 Effects of sugar content of liquid media on the plating
efﬁciency and colony formation in bead culture ............... 30

1.3 Effects of hormone concentrations of liquid media on

plating efﬁciency and colony formation in head culture ........... 33
1.4 Effects of liquid, agarose layer and bead type

cultures on plating efﬁciency and colony formation ............. 34
2.1 Source of twenty primers used to screen the resistant

somaclones R7, R4 and four control plants .................. 55

2.2 Analysis of variance of a randomized complete block
design for Fusarium spp. as factor A and germplasm
entries as factor B, which is a split plot on A. ................ 62

2.3 Mean disease rating of asparagus plants screened with
Fusarium oxysporum (isolates FOAlO and FOASO) and F.
proliferatum (isolates FM12 and FM 49) in the greenhouse ........ 63

2.4 Disease rating of asparagus plants screened with
Fusan’um oxysporum (isolates FOAlO and FOASO) and F.
proliferatum (isolates FM12 and FM49) in the greenhouse ......... 64

2.5 Disease rating of vegetatively micropropagated plants
of the more resistant somaclone R7 rescreened with
Fusarium oxysporum (isolate FOASO) in the greenhouse .......... 66

2.6 Disease rating of vegetatively micropropagated plants
of the more resistant somaclone R4 rescreened with
Fusarium oxysporum (isolate FOASO) in the greenhouse .......... 67

TABLE PAGE

2.7 Number of unique ampliﬁcation products that were all present
in the resistant somaclone R7 , and present (P) or absent (A) in
the resistant somaclone R4 when compared to 4 control plants ...... 75

A.1 Detection and quantitation of GUS activity of putative transgenic
plants (PTP) and non-transformed plants by ﬂuorometric assay ...... 96

A.2 Detection and quantitation of NPTII protein in crude cellular
extracts of transgenic and non-transgenic plants by NPTII Elisa ...... 98

A3 Inhibition of E. coli growth by crude
protein extracts from transgenic plant T11 ................... 101

A.4 Disease rating of vegetatively micropropagated plants of transgenic
plant T11 and nontransgenic plants, and control plants screened
with Fusarium oxysporum (isolate F OA50) in the greenhouse ....... 103

xi

LIST OF FIGURES

FIGURE PAGE
1.1 Development of A. oﬂicinalis cv. Lucullus 234 from

callus-derived protoplasts to colony in bead culture (a-d) .......... 28
1.2 Development of A. oﬁ‘icinalis cv. Lucullus 234 from colony to plant (a-d) 29
1.3 Effect of cytokinin type and concentration, in combination

with auxins in the shoot-inducing medium of protoplast-

derived calli of asparagus, on shoot regeneration ( %) ............ 37
1.4 Asparagus plants from protoplasts growing in the greenhouse ....... 38
2.1 Effects of different auxins (1AA, IBA, NAA, and 2,4—D on

rooting frequency (96) of the shoots produced on RM17 medium

of asparagus cv. Lucullus 234 after 4 weeks in culture ........... 58
2.2 Effects of IBA concentrations on rooting frequency

(96) of the shoots produced on RM17 medium of

asparagus cv. Lucullus 234 after 4 weeks in culture ............. 59
2.3 Four-week-old plantlets of asparagus cv. Lucullus 234 in

RTM26 medium containing basic MS medium + 3 mg/l IBA ....... 60
2.4 The chromosomes in A. oﬁcinalis cv. Lucullus 234 (2n =20) ....... 69
2.5 The chromosomes in the resistant somaclone R7 of

A. oﬁ‘icinalis cv. Lucullus 234 (2n = 20) ................... 70
2.6 The chromosomes in the resistant somaclone R4 of

A. oﬁicinalis cv. Lucullus 234 (2n = 20) ................... 71
2.7 Unique polymorphisms found in the resistant somaclone R7 when

compared to ’Lucullus 234’ parental plants (primers from B to L) . . . . 73
2. 8 Unique polymorphisms found in the resistant somaclone R7 when

compared to ’Lucullus 234’ parental plants (primers from M to T) . . . . 74

xii
FIGURE PAGE

2.9 Unique polymorphisms found in the resistant somaclone
R4 when compared to ’Lucullus 234’ parental plants ............ 76

A.1 Restriction map of the T—DNA region of
pB1121 plasmid in A. tumefaciens LBA4404 ................. 89

A.2 Autoradiogram of Southern blot of DNA extracted from transformed
and untransformed plants, digested with restriction enzyme HindIII
and membrane hybridized with 1.7 kb Shiva-l probe ............ 99

NAA
2,4-D
6—BAP
IBA
IAA

kb

LIST OF ABBREVIATIONS

alpha-naphthaleneacetic acid
2,4—dichlorophenoxyacetic acid
6-benzylaminopurine
indolebutyric acid

indoleacetic acid

kilobases

INTRODUCTION

Botanical aspect

The genus Asparagus, a member of the family Liliaceae, has about 150-300
herbaceous and woody perennial species widely grown in the temperate and tropical
regions of the world (Lawrence, 1982). Asparagus oﬁ‘icinalis L. is native to the Orient
and to the east of the Mediterranean genecenter, and has been the only species cultivated
as an edible vegetable plant for over 2,000 years. A. oﬁ‘icinalis is dioecious with 2n =
20 (Jessop, 1966) and a sex ratio of 1:1; however, male plants have both staminate and
hermaphroditic ﬂowers (Reuther, 1984). Female plants are homogametic XX, male
plants are heterogametic XY, and hermaphroditic ﬂowers arise on male (XY) plants due
to a rare mutation. The male with hermaphroditic ﬂowers are andromonoecious plants
which can be cross- or self-fertilized (Wircke, 1979; Reuther, 1984). Andromonoecious
plants produce two genotypes: XY male and YY super-male after self-fertilizing.
Crossing supermales with females produces all-male plants which are most desirable
because of their higher yield, vigor, and longevity (Ellison et al. , 1960). In addition,
increase of disease resistance and drought tolerance are associated with male plants
(Ellison, 1986). Andromonoecious plants can make up 10 to 20% of some asparagus

populations (Wircke, 1979).

2
Importance and problems of asparagus production

A. oﬁ'icinalis is an economically important vegetable crop in over 17 countries
(Reuther, 1984). The United States produces 45,870 ha with California (17,180 ha),
Washington (13,640 ha), Michigan (9,320 ha), and New Jersey (870 ha) as the primary
producers, and is the most important country in the world for asparagus production
(Desjardins, 1992). Asparagus is one of the most important vegetable crops in Michigan
(Kindinger, 1987) with an average annual value of over 15 million dollars (Fedewa and
Pscodna, 1993). It is a perennial vegetable crop, and properly maintained asparagus
ﬁelds should remain productive for 20 or more years with a yield of 2-3,000 lb per acre
or more. However, today’s asparagus ﬁelds are being removed from production after
only 8 to 15 years due to sparse stands and small spear size, resulting in low yields
(Takatori and Souther, 1978). This problem is due to a disease syndrome known as
”asparagus decline” which causes loss in longevity and productivity of established ﬁelds,
difﬁculty in replanting asparagus where asparagus was previously grown, and thereby
decreases in annual yields of asparagus over time. The disease is found throughout the
world (Cohen and Heald, 1941; Graham, 1955; Grogan and Kimble, 1959; Van Bakel
and Kerstens, 1970; Endo and Burkholder, 1971). Most ﬁeld are planted with about
10,000 crowns per acre, but often 50% are lost in the ﬁrst ﬁve years after planting due
to ”asparagus decline”. In Michigan asparagus ﬁelds, the average crown population was
3153 per acre from 1978 to 1979, and this represented a 70% reduction in crown
survival (Hodupp, 1983). In 1978, the average yield for Michigan was 1,5001b/A; but
in 1981 the state average was only 9001b/A (Bates et al. , 1987). Asparagus decline also

reduced the acres cultivated. Asparagus production has decreased from 30,000 acres to

3
less than 1000 over the last 25 years in New Jersey (Hemer and Vest, 1974), from

44,000 acres in 1974 to 28,000 in 1978 in California (Takatori and Souther, 1978) and

from 500 ha in 1963 to 340 in 1970 in the Netherlands (Van bake] and Kerstens, 1970).

Factors contributed to the problems

Abiotic factors such as environmental stresses (nutrient imbalance, low soil pH and
soil moisture), physical stresses (defoliation by asparagus beetle, culture practice and
allelopathic substances produced by asparagus tissue), and biotic factors such as
asparagus virus I and II, and Stemphylium vesicarium have been implicated in the decline
(Kitahura et al., 1972; Laufer and Garrison, 1977; Yang, 1982, 1985; Falloon et al.,
1984; Young, 1984; Evans and Stephens, 1985; Shafer and Garrison, 1986).

However, the most important factors associated with "asparagus decline" are
Fusarium axysporum f. sp. asparagi Cohen and Heald and F. proliferatum (T.
Matsushima) Nirenberg (syn. F. monilrfomte J. Sheld.)(Cohen and Heald, 1941; Graham,
1955; Grogan and Kimble, 1959; Van Bakel and Kerstens, 1970; Endo and Burkholder,
1971). F. oxysporum and F. proliferatum are important pathogens of major crops
throughout the world. They are chitinous and facultative parasites which colonize living
and non-living host tissue and may invade non-host tissue (Alexander, 1961; Hendrix et
al. , 195 8). They are able to form chlamydospores or other resting structures which can
survive in soil for many years. These characteristics make them especially persistent
once they are established (Nelson et al., 1981). F. oxyspomm causes a vascular wilt
which inhibits water uptake and carbohydrate transport within the plant. F. proliferatum

is primarily a root-rotting organism. Asparagus plants infected by either pathogen show

4
similar above-ground symptoms; the ferns are yellowed, stunted, wilted, and may die in

different stages of development (Cohen, 1946; Endo and Burkholder, 1971). F.
oxysporum eauses root rotting and the symptoms of vascular discoloration within the
stem, root and crown (Endo and Burkholder, 1971). F. proliferatum causes more
extensive dry crown rot and brown stem pith discoloration but no vascular discoloration

(Cohen, 1946).

Conventional controb of Fusarium and their limitations

Conventional methods of controlling Fusarium spp. are limited. Chemical
treatments, including crown dips and foliar sprays, have not been successful (Stephens
et al. , 1991). Funrigation does not offer long-term effectiveness because of the perennial
nature of the crop (Lacy, 1979). Further, Fusarium spp. are ubiquitous and may reinfest
the soil following fumigation and rapidly colonize young asparagus plants in the ﬁeld
(Damicone and Manning, 1985). Cultural control such as incorporation of cruciferous
residues reduces Fusarium population, but does not eliminate the disease (Stephens and
Sink, 1992). To date, no Fusarium resistant varieties have been developed (Takatori and

Souther, 1978; Ellison, 1986).

Alternative strategies to control Fusarium

The most successful strategy for Fusarium wilt control in other vegetable crops has
been the development of resistant varieties (Mace et al. , 1981). Possibilities exist for
developing Fusarium resistant varieties in asparagus. A cultivar of A. oﬁ‘icinalis,

Lucullus 234, had the highest resistance to virulent Michigan isolates FOAlO of F.

5
oxysporum and FM12 of F. proliferatum among 90 cultivars and breeding lines of this

species tested (Stephens et al. , 1989). Two ornamental cultivars of Asparagus
densiﬂoms (Kunth) J cssop (cvs. Sprengcri and Myersii) were resistant to F. oxysporum
and F. pmliferatum in greenhouse studies (Stephens et al. , 1989). Further, it was
suggested that A. densiﬂorus cv. Sprengcri was immune to F. oxysporum f. asparagi in
a laboratory study (Lewis and Shoemaker, 1964). However, sexual crosses of A.
oﬂ'icinalis with the resistant species, A. densiﬂorus ’Sprengeri’ have been unsuccessful,
probably due to incompatibility barriers (Elmer et al. , 1989). In addition, using
conventional methods, A. oﬁ'icinalis has low regenerative potential (Reuther, 1984), and
the development of new cultivars requires many years because of its perennial nature.
It is likely that genetic diversity among the asparagus cultivars in North America is low
(Gleason and Cronquist, 1963; Luzny, 1979; Ellison, 1986). In this context, tissue
culture and micropropagation were investigated as a possible means of locating resistant
lines.

Isolated protoplasts are a unique tool for genetic manipulation of plants such as
regeneration of the entire plant from protoplasts, somatic fusion between sexually
incompatible plant species and incorporation of interesting DNA into protoplasts. Plant
regeneration from protoplasts has enabled investigators to create new protoplast-derived
somaclones as novel genetic sources. Protoplast fusion has provided somatic
hybrids/cybrids at interspeciﬁc and intergeneric levels for widening the pools of
germplasm in crop improvement programs. In addition, the storage of protoplasts
through immobilization and cryopreservation are of great importance, especially for the

pharmaceutical industry (Bajaj , 1989). Since the ﬁrst report on the regeneration of

6
complete plants from protoplasts (Takebe et al. , 1971), remarkable progress has been

made and a number of commercially important crops such as potato, tobacco, tomato,
rice, maize, cucumber and eggplant etc. are routinely regenerated (Bajaj, 1989).
Genetic variability was ﬁrst reported in cultured plant cells in 1967 (Murashige and
Nakano, 1967). Variation among plants regenerated from tissue culture has been termed
”somaclonal variation" (Larkin and Scowcroft, 1981). Variation has been detected in
cultured cells, especially on periodical subculturing for various morphological and genetic
changes, such as polyploidy, aneuploidy, chromosome breakage, deletion, translocation,
gene ampliﬁcation, inversions, mutations, etc. (Nagl, 1972; Meins, 1983; D’Amato,
1985). Molecular and biochemical changes occurred in the DNA (Berlyn,1982; Cullis,
1983), enzymes (Brettell et al., 1986), gliadin (Cooper et al. , 1986), etc.. Somaclonal
variation was ﬁrst proposed as a novel source of agriculturally useful variation for
asexually propagated crops such as sugarcane (Heinz et al. , 1977) and potato (Shepard
et al. , 1980). Somaclonal variation has been used to recover genetic variability at high
frequency in a number of crop varieties, and offers an alternative to mutation breeding.
Somaclones have shown resistance! tolerance to pathogenic fungi (Forougi-Wehr et al. ,
1986; Rines, 1986; Meulemans et al. , 1987), bacteria (Thanutong et al., 1983;
Hammerschalg, 1986; Sun et al., 1986), viruses and nematodes (Wenzel and Uhrig,
1981), phytotoxins (Gengenbach et al. , 1977), herbicides (Chaleff and Ray, 1984), and
salt (Nabors et al., 1980; Bajaj and Gupta, 1987). In addition, high-yielding (Ogura et
al. , 1988), rich in protein (Schaeffer and Sharpe, 1987) and sugar contents (Liu and
'Chen, 1976), and male sterility (Ling er al. , 1987) have been obtained in somaclones of

different crops. Important agricultural crops such as wheat, rice, maize, potato,

7

sugareane, brassica. etc. have already yielded positive results to the extent of new
cultivars being released (Bajaj , 1990).

The soil bacterium, Agrobacterium tumefaciens, can to infect most dicotyledonous
plants at wounding sites (Braun, 1978, 1982) and induce formation of tumors, called
crown gall, via the transfer of a plasmid it contains, called the Ti-plasmid (Zaenen et al. ,
1974; Van Larebeke et al. , 1974; Watson et al. , 1975). When the bacterium comes in
contact with a wounded plant cell, the Ti—plasmid is transferred from the bacterium into
the cell. A small segment of the plasmid, T-DNA, is transferred from the plasmid to the
nucleus of the plant cell, and becomes integrated into the plant nuclear genome (Chilton
er al. , 1977, 1978; Willmitzer et al. , 1980). This organism became the main target for
development of a plant transformation technique. Agrobacterium—mediated gene transfer
is now a powerful tool for introducing foreign genes into many plant species to improve
agricultural crops by increasing of resistance to pathogenic viruses (Nelson at al. , 1988;
Hoekema et al. , 1989), fungi (Broglie er al., 1991) and bacteria (Destefano-Beltran et
al. , 1990). Traditionally it has been considered that Agrobacterium—mediated
transformation was limited to dicotyledonous plant species; however it is now becoming
increasingly clear that Agrobacten'wn can transfer DNA to cells of monocotyledonous
plants such as asparagus (I-Iernalsteens er al. , 1984; Bytebier et al. , 1987; Delbreil et al. ,
1993), rice (Raineri et al., 1990), wheat (Mooney et al. , 1991) and corn (Graves and
Goldman, 1986).

It was possible that highly disease resistant asparagus cultivars could be derived from
selection of somaclonal variation among plants regenerated from protoplasts of ’Lucullus

234’ in response to F. oxysporum and F. proliferatum, Agrobacterium—mediated or direct

8
gene transfer of asparagus with foreign fungal resistant genes such as Shiva-l gene, and

protoplast fusion of ’Lucullus 234’ with the resistant A. densiﬂorus ’Sprengeri’ or

’Myersii’ .

Objectives of this study

The overall objective of these studies was to develop Fusarium resistance of
asparagus via tissue cultures and gene transformation, and determine the genetic base of
the resistance. To approach this goal, the following studies were conducted 1) to develop
a procedure to isolate, culture and regenerate plants from callus-derived protOplasts of
’Lucullus 234’ , 2) to develop protocols for vegetative micropropagation and somaclone
production of ’Lucullus 234’ , 3) to screen protoplast-derived somaclones of ’Lucullus
234’ for resistance to F. oxysporum and F. proliferation, and characterize the genetic
base of the Fusarium resistant somaclones by randomly ampliﬁed polymorphic DNAs
(RAPDs) technology and cytogenetic study, and 4) to develop Agrobacterium-mediated
gene transformation of asparagus (’Lucullus 234’) with antifungal gene ’Shiva-l ’, verify
the transformed plants and evaluate the transformed plants in response to the most

virulent Michigan isolate of F. oxysporum, FOA50.

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Bajaj,Y.P.S. 1989. Recent advances in the isolation and culture of protoplasts and their
implications in crop improvement, pp. 3-5 . In: Biotechnology in agriculture and forestry
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Bajaj ,Y.P.S. 1990. Somaclonal variation - origin, induction, cryopreservation, and
implications in plant bmding, pp. 3-49. In: Biotechnology in agriculture and forestry
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Bates,G.W., Hasenkampf,C.A., Contolini,C.L. and Piastuch,W.C. 1987. Asymmetric
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Berlyn,M.B. 1982. Variation in nuclear DNA content of isonicotinic acid hydrazide-
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Boller,T., Gehri,F., Mauch,F. and Vogeli,U. 1983. Planta 157:22.
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Brettell,R.I.S., Dennis,E.S., Scowcroft,W.R. and Peacock,J.W. 1986. Molecular
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CHAPTERI

Optimal Conditions for Growth and Plant Regeneration of Asparagus (Asparagus

Oﬂiciualis L. cv. Lucullus 234) from Callus-Derived Protoplasts

ABSTRACT

Efﬁcient protocol was developed for plant regeneration from callus-derived
protoplasts of Asparagus oﬁicinalis L. cv. Lucullus 234. In addition, the effects of
protoplast culture media on protoplast growth were investigated as well as types of
culture, and osmotic conditions or hormone concentrations in liquid media of bead
culture. Plating efﬁciency and colony formation differed signiﬁcantly among different
protoplast culture media and types of culture. Osmotic conditions and hormone
concentrations of liquid media had the greatest inﬂuence on plating efﬁciency and colony
formation in bead culture. Protoplasts grew best in bead culture with a solid modiﬁed
Kao & Michayluk protoplast culture medium (KM) supplemented with 0.5 mg l'1 N AA,
0.5 mg l'1 2,4-D, 0.5 mg l‘1 kinetin, and 0.6% agarose (KM6), and in a similar liquid
medium differing in sugar content, having 0.18 M sucrose and 0.18 M mannitol (A8).
An average plating efﬁciency and colony formation (19.1% and 15.5 % respectively) was
obtained one week after isolation in bead culture with the KM6 and A8 media. The

highest average shoot regeneration of 92.3 % was obtained with a Murashige & Skoog

17

18
medium (MS) containing 0.125 mg 1-1 NAA, 0.125 mg 1-1 2,44), 0.25 mg 1'1 amp and

3 % sucrose. Plants have been regenerated after shoots were transferred into hormone-

free MS medium. Plants were transplanted in the greenhouse.

INTRODUCTION

Asparagus (Asparagus oﬁ‘icinalis L.) is an important vegetable crop and produced
worldwide (Desjardins, 1992). However, a disease syndrome known as asparagus
decline, primarily caused by Fusarium oxysporum f. sp. asparagi Cohen and Heald and
F. proliferation (T. Matsushima) Nirenberg, decreases annual yields of asparagus over
time, and is found throughout the world (Cohen and Heald, 1941; Graham, 1955; Grogan
and Kimble, 1959; Van Bakel and Kerstens, 1970; Endo and Burkholder, 1971).
Attempts to sexually cross A. oﬂicinalis with the resistant ornamental species, A.
densrﬂoms ’Sprengeri’ or ’Myersii’ have been unsuccessful, probably due to
incompatibility barriers (Elmer et al. , 1989). Also, chemical and cultural controls of
Fusarium spp. in asparagus ﬁelds have not been effective (Lacy, 1979; Stephens et al.,
1991; Stephens and Sink, 1992). Tissue culture techniques offer new approaches to
develop Fusarium resistant asparagus from a cultivar of A. oﬁ‘icinalis, Lucullus 234,
which had the highest tolerance to F. oxysporum and F. proliferation compared to 90
cultivars and breeding lines of this species (Stephens et al. , 1989). Protoplast fusion
provide the only current means of obtaining somatic hybrid plants to overcome sexual
incompatibility barriers . Protoplasts have been used for direct uptake of desired DNA
or chromosomes by chemically mediated, electroporation, and microinjection methods

into target plants (Lal, 1990). In addition, plant regeneration from protoplasts offers

19

novel somaclonal variation for crop improvement such as increase of disease resistance
(Bajaj , 1990)). However, to pursue these approachs, an efﬁcient protoplast regeneration
protocol is necessary.

The division of protoplasts from Asparagus oﬁicinalis and their growth and
differentiation were reported by Bui Dang Ha and Mackenzie (1973). The same group
regenerated plants of A. oﬁ'icinalis through callus cultures derived from protoplasts (Bui
Dang Ha and al., 1975). Chin et al. (1988)reported culture of droplets containing
asparagus cells and protoplasts on polypropylene. Kong and Chin (1988) regenerated
plants of A. oﬁ'icinalis using agarose on polypropylene membrane. Elmer et al. (1989)
reported plant regeneration from callus-derived protoplasts of A. oﬁicinalis. Plating
efﬁciency (the numbers of cells divided / total protoplasts plated) was low, and cell
division, colony formation and callus formation were slow in the above studies of
protoplast isolation, culture and plant regeneration of different cultivars of A. oﬁ'z‘cinalis.
In addition, only three of these investigations reported plant regeneration (Bui Dang Ha
et al., 1975; Kong and Chin, 1988; Elmer et al., 1989). Bead culture is used
successfully in protoplast culture of other plant species to increase plating efﬁciency
(Shillito et al. , 1983; Furze et al., 1987), but has not been used in protoplast culture for
asparagus. Further, the effects of osmotic conditions and hormone concentration in
liquid media in bead culture on protoplast growth have rarely been reported.

This chapter reports the development of a new protocol for culture, isolation and
plant regeneration of callus-derived protoplasts of asparagus cultivar Lucullus 234. In
addition, the objectives were to study the effects of : 1) liquid, argrose-layer and bead

culture on plating efﬁciency and colony formation of protoplasts; 2) different types (2,4-

20

D, NAA, kinetin, zeatin and 6-BPA) and concentrations of plant growth regulators on
plating efﬁciency and colony formation; 3) osmotic conditions and hormone
concentrations in liquid medium of bead culture on plating efﬁciency and colony

formation; 4) different cytokinins (6-BAP, zeatin and kinetin) on shoot differentiation.

MATERIALS AND METHODS

Plant material Shoots from 2- to 3-year old greenhouse-grown plants of A. oﬁ‘icinalis
cv. Lucullus 234 were surface-sterilized for 30 m in a 20% (v/v) aqueous bleach solution
(5.25 % sodium hypochlorite, Big Chief Bleach, Patterson laboratories, Detroit MI) plus
3 ml per liter of Tween-20 (Sigma Chemical Co., St. Louis MO). Shoots were rinsed
three times with sterile distilled water and placed in 100 x 15 mm plastic Petri dishes
containing MSF medium (Elmer et al., 1989). After incubation in the dark at 27°C for

one month, calli were excised from the shoots and subcultured every four weeks.

Isolation of protoplasts Callus (2.5 g) was removed from subculture, sliced thinly with
a new scalpel blade, and placed in 30 ml of enzyme solution (Elmer et al. , 1989) of 1%
Ccllulysin, 0.2% Macerase and 1% Rhozyme dissolved in CPW solution (Cooking and
Peberdy, 1974) at pH 5.6 sterilized by passing through a 0.22pm ﬁlter. Protoplasts were
released during a 16 to 17 h digestion period in the dark at 27 to 28°C on a gyratory
shaker at 30 rpm. Tire protoplasts were puriﬁed using the modiﬁed protocol of Elmer
et al. (1989). Protoplasts were collected through a 61 um nylon sieve separating

undigested clumps of callus. Protoplasts were washed three times with CPW solution

21
plus 0.7 M mannitol at pH 5.85 (CPW wash solution) and centrifuged at 70 g for 5 m.

Protoplasts were ﬂoated in CPW solution with 21% sucrose (pH 5.85) on top of which
3 ml of CPW wash solution was layered and centrifuged at 150 g for 5 m. After
centrifugation, protoplasts were separated from undigested cells at the interface between
the solutions. Protoplasts were collected, suspended in CPW wash solution, counted with
a hemacytometer, centrifuged at 70 g for 5 m and diluted to 5 x 104 ml'1 for culturing

in different protoplast culture media.

Culture of protoplasts The isolated protoplasts were cultured at a density of 5 x 104
ml'1 . Several factors were tested for their effects on plating efﬁciency and colony
formation: types of culture (liquid, agarose layer, and bead cultures); growth regulators
for liquid and agarose layer cultures; and osmotic condition and hormone concentrations
of liquid media of bead culture.

Six different liquid protoplast media were tested for culturing in liquid or on agarose
layer cultures. These media were based on a modiﬁed Kao & Michayluk protoplast
culture medium (KM) containing 0.05 M sucrose, 0.05 M glucose and 0.7 M mannitol
(Elmer et al., 1989). Hormone content of the six media were (mg 1"): KM1= 2,4-D,
1 + 6-BAP, 0.5; KM2= 2,4-D, 1 + kinetin, 0.5; KM3= NAA, 1 + 6—BAP, 0.5;
KM4= NAA, 1 + kinetin, 0.5; KMS= NAA, 0.5 + 2,4-D, 0.5 + 6-BAP, 0.5, and
KM6= NAA, 0.5 + 2,4-D, 0.5 + kinetin, 0.5. Liquid cultures had 3 ml of protoplast
suspension in liquid media plated onto 60 x 15 mm plastic Petri dishes. Agarose layer
cultures had 3 ml of protoplast suspension in liquid media over 3 ml of the same media

solidiﬁed with 0.4% agarose (Sea Plaque LMT, FMC Corp., Rockland ME). At weekly

22
intervals, 0.5 ml of the initial liquid protoplast culture media was removed and

substituted with 0.5 ml of replacement media. Replacement media differed from the
original media in sugar content (0.18 M sucrose and 0.18 M mannitol) and did not
contain auxins. The plates were incubated at 27°C in the dark for 1 week followed by
1 week under 2 to 3 rtEm'zs'l light with 16 h photoperiod. The plates were maintained
for an additional 3 weeks at 27°C with 40 pEm'zs'l light and a 16 h photoperiod. All
light was provided by cool-white ﬂuorescent lamps.

For studies on effects of osmotic conditions of liquid medium in bead culture, KM6
was chosen as the solid medium in which 0.6% agarose was added for bead culture.
Osmotic conditions of liquid medium in bead culture affected protoplast growth. This
was investigated by comparing A8 liquid medium to KM6 liquid medium (control). A8
liquid medium differed from liquid medium KM6 (800 mOsmol/kg H20) in sugar
content, having 0.18 M sucrose + 0.18 M mannitol (360 mOsmol/kg H20). The bead
cultures were prepared according to the method of Shillito et al. (1983). Protoplasts
were suspended in molten KM6 solid medium and 4 ml was plated onto 60 x 15 mm
plastic Petri dishes. After solidiﬁcation of the agarose (25-30 min), the agarose was then
cut into 6 pieces and transferred to 30 ml of A8 and KM6 liquid medium in 100 x 20
mm plastic Petri dishes. Ten ml of initial liquid protoplast culture medium were
removed and replaced with 10 ml of original fresh liquid medium every week for all bead
cultures. The plates were placed on a gyratory shaker (55 rpm) at 27 °C in the dark.

To study effects of hormone concentration of liquid medium of the bead culture on
protoplast growth, A8 liquid medium was chosen because it was found have better plating

efﬁciency than KM6 liquid medium for bead culture. KM6 medium was used as the

23
solid medium. Then, three liquid media similar to A8 medium but differing in growth

regulator concentration were tested in bead culture. The three media were: A7,
containing NAA, 2,4-D and kinetin at concentrations of 0.25 mg 1'1; A8, containing
NAA, 2,4-D and kinetin at concentration of 0.5 mg 1“, and A9, containing NAA, 2,4-D
and kinetin at concentration of 1.0 mg 1". The bead cultures were prepared and
maintained using the methods described previously.

For comparison of liquid, agarose layer, and bead culture, KM6 medium was
selected as the protoplast culture medium for all cultures, and A8 liquid medium was
used as both liquid medium for bead culture and replacement medium for all cultures.
These three cultures were prepared using the methods described previously. At weekly
intervals, 1 ml (for the liquid and agarose layer) and 10 ml (for bead culture) of the
initial liquid protoplast culture medium were removed and replaced respectively with 1
ml and 10 ml of the fresh A8 liquid medium. The plates of bead culture were placed on
a gyratory shaker (55 rpm) at 27°C in dark. The plates of liquid and agarose layer
cultures were incubated at 27 °C in dark.

Plating efﬁciency, is deﬁned as the numbers of cells divided / total protoplasts
plated. Colony formation, is deﬁned as the numbers of cell colonies of more than three
cells / total protoplasts plated. Plating efﬁciency and colony formation were recorded
one week after isolation for bead culture. Plating efﬁciency was evaluated three weeks
after isolation and colony formation recorded ﬁve weeks after isolation for liquid and
agarose layer cultures. At least 500 cells of more than ﬁve random microscopic ﬁelds
were examined. Protoplast yield is deﬁned as the numbers of protoplasts per gram of

callus. Protoplast yield was counted after ﬂotation and the ﬁnal wash (before plating in

24

protoplast culture media). All experiments were conducted as a completely randomized
design with six replicate plates per treatment and were conducted at least three times.
Values represent the mean of six replicate plates from at least three isolations. All the
data in the tables were analyzed by ANOVA and Duncan’s Multiple Range Test was used

to separate means.

Plant regeneration of calli from single protoplasts Two to four weeks after initiation
of the bead culture, calli formed from protoplasts were transferred to nine different
shoot-inducing media and a hormone-free medium (control). The nine shoot-inducing
media were made from three combinations of cytokinins and auxins at three
concentrations. All media were based on Murashige & Skoog (MS) medium (1962) +
30 gl'1 sucrose at pH 5.85. The three hormone combinations were NAA:2,4-D:Kinetin,
NAA:2,4-D:Zeatin, and NAA:2,4-Dz6-BAP. Each hormone combination was at three
concentrations of (mg 1") o.125:o.125:o.25 (level 1), o.25:o.25:1 (level 2), and cm
(level 3). MS medium without hormones was prepared as control. The plates were
incubated at 27°C in the dark for three weeks, under 2 to 3 llamas-1 for one week and
40 pEm'zs’1 with 16 h photoperiod for 1 week. Four weeks later, calli were transferred
from shoot-inducing media to hormone-free media for shoot and plant regeneration at
27 °C with 40 trEm'zs'1 light and a 16 h photoperiod. Plants were transferred to the
greenhouse after they regenerated directly in hormone-free medium. All experiments for
shoot induction contained at least six replicates (6 to 12 calli per Petri dish) per treatment

and were conducted three times.

25
RESULTS AND DISCUSSION

Effects of different media on plating efficiency and colony formation in liquid and
agarose layer cultures An average of protoplast yields from all the isolations was 1.23
x 106 prot0p1asts / gram of callus. Isolated protoplasts (Figure 1. la) became
nonspherical within 24 h after plating as their cell walls reformed. Two to nine percent
of the surviving cells became oval and some budding occurred within 1 week. Three to
seven days after plating, some cells entered into the ﬁrst cell division, but did not
continue to grow in liquid or agarose layer cultures. The ﬁrst colony of four to six cells
appeared 4 weeks after isolation. Both media and type of culture, and the interaction had
a signiﬁcant effect on plating efﬁciency (ANOVA, P<0.05). KM6 or KM5 media
combined with agarose layer culture signiﬁcantly increased plating efﬁciency over other
media and culture types (Table 1.1). Only media signiﬁcantly inﬂuenced colony
formation. The type of culture and the interaction between media and type of culture did
not signiﬁcantly affect colony formation. The colony formation was signiﬁcantly higher
with KM6 medium combined with agarose layer culture in comparison to other media
(Table 1.1). Hormone content and agarose in the protoplast media played an essential
role in protoplast culture. By the combination with agarose layer culture, only the media
in the presence of NAA (KM6 = KM basal + 0.5 mg NAA + 0.5 mg 2,4-D + 0.5 mg
kinetin/l and KM5 = KM basal + 0.5 mg NAA + 0.5 mg 2,4-D + 0.5 mg 6-BAP/l)
signiﬁcantly increased cell division. KM6 stimulated sustained division. This
observation is in contrast to Elmer at al. (1989), who obtained sustained division of

asparagus prot0p1asts only in a similar medium without NAA (the same KM basal +

26

Table 1.1. Effects of different media and types of culture on plating efﬁciency and
colony formation of asparagus protoplasts.

 

Protoplast Type of culture Plating efﬁciency (%)l Colony formation (%)1

 

 

 

 

 

 

medium (cells divided / total (colonies > 3 cells /
protoplasts plated) total protoplasts plated )
Agarose layer 1. 38 A 1 .27 A
KM 6 , ,
quurd 0.29 C 0.43 B
Agarose layer 0.91 AB 0.41 B
KM 5 , ,
quurd 0.00 C 0.00 B
Agarose layer 0.49 BC 0.00 B
KM 4 , ,
quurd 0.18 C 0.00 B
Agarose layer 0.24 C 0.00 B
KM 3 , ,
quurd 0.14 C 0.00 B
Agarose layer 0.14 C 0.07 B
KM 2 , ,
quurd 0.14 C 0.00 B
Agarose layer 0.06 C 0.00 B
KMl , .
quurd 0.03 C 0.00 B

 

1Means of 6 replicate plates from all four isolations. Plating efﬁciency was evaluated
3 weeks after isolation and colony formation was recorded 5 weeks after isolation.
Means within a column with different letters are signiﬁcantly different (Duncan’s
multiple range test, P < 0.05).

27
1mg 2,4—D + 0.5 mg 6-BAP/l). Elmer et al. concluded that NAA was not required for

sustained division of asparagus protoplast, but they used different cultivars. However,
the plating efﬁciencies (0.0% to 1.4%) and colony formation (0.0% to 1.3%) were very

low, and few calli were obtained after 8 weeks of culture for all treatments investigated.

Effects of osmotic condition of liquid media on plating efficiency and colony
formation in head culture The ﬁrst cell division occurred the second day after isolation
(Figure 1.1b), and colony formation with four to twelve cells appeared after 3 days in
the liquid medium A8 in bead culture (Figure 1.10). Cell colonies grew up to 0.48 mm
diam. (Figure 1.1d) within 1 week and developed into microcalli (1 mm diam.) within
2 weeks after isolation (Figure 1.2a). Average plating efﬁciency and colony formation
of A8 liquid medium (19.1% and 15.5% respectively) was signiﬁcantly greater than
those of KM6 liquid medium (control) (1.1% and 0.2% respectively)(Table 1.2). With
KM6 medium as the liquid medium (control) in the bead culture, cell division began 2
to 4 days after isolation and colony formation occurred 1 week after isolation. Although
a few divided cells and colonies were present 1 week after isolation, they did not
continue to grow and did not form calli (Table 1.2). In addition, after bead cultures
were maintaining in KM6 liquid medium for 3, 5 and 7 days, the KM6 liquid medium
were totally replaced by 30 ml of A8 medium. The protoplasts did not continue to grow
and died although a few cell divided. The high osmotic condition of KM6, when used
as the liquid medium of bead culture, signiﬁcantly decreased the plating efﬁciency and
colony formation, and completely inhibited any subsequent callus formation (Table 1.2).

Although the liquid A8 medium had 0.18 M mannitol and 0.18 M sucrose without

28

 

 

 

Figure 1.1. Development of A. oﬂicinalis cv. Lucullus 234 from callus-derived
protoplasts to colony in bead culture (a-d). (a) Asparagus protoplasts immediately after
isolation from calli cultured in medium MSF; (b) ﬁrst division of protoplasts embedded
in agarose of head culture 1 day after isolation; (c) a colony of about 10 cells derived
from protoplasts embedded in agarose of bead culture 5 days after isolation; (d) a cell
colony derived from protoplasts embedded in agarose of bead culture 1 week after
isolation.

29

 

Figure 1.2. Development of A. oﬁ'icinalis cv. Lucullus 234 from colony to plants (a-d).
(a) microcalli derived from protoplasts embedded in agarose of bead culture 2 weeks
after isolation; (b) calli derived from protoplasts on shoot-inducing medium with 6-BAP
at level 1 one week after initiation of bead culture; (c) callus regenerated from
protoplasts with shoots and plantlets in hormone-free medium; (d) plant regeneration
from protoplast-derived calli in hormone-free medium.

30

Table 1.2. Effects of sugar content of liquid media on the plating efﬁciency and colony

formation in bead culture.

 

 

 

Liquid Osmolarity Plating Colony formation l‘hcell Colony Microcalli
media (mOsmol/kg efﬁciency (96)1 (96)1 (colonies > division formation formation
H20) (cell divided / 3 cent / total (days) (days) (days)
total protoplast protoplasts plated)
plated)
A8 360 19.12 A 15.52 A 2 3 10
KM6 800 1.08 B 0.19 B 2-4 7 no
microcalli

 

1Means of 6 replicate plates from all three isolations. Means within a column with

different letters are signiﬁcantly different (ANOVA, P < 0.05). Plating efﬁciency and

colony formation was evaluated 1 week after isolation.

31

glucose, the presence of glucose (0.05 M) in liquid KM6 medium was not considered to
have inhibitory effect on protoplast growth because amount of glucose was only 6% of
total sugar in the medium. Also glucose is widely used in protoplast culture including
asparagus protoplast culture and may be the preferred carbon source for most protoplasts
(Evans and Bravo, 1983; Elmer et al., 1989). These results indicate that the high
osmotic conditions, which is considered to be necessary to prevent bursting of protoplasts
during early culture (Evans and Bravo, 1983), inhibited cell division later and eventually
result in cell death. Bead culture permits use of lower concentrations of sugar in the
liquid medium, thereby reducing the osmotic conditions immediately after plating the
protoplasts without bursting protoplasts. High osmotic pressure impairs metabolism and
growth of protoplasts (Evans and Bravo, 1983). This can cause reduced uptake of amino
acids across the plasma membrane (Reusink, 1978) and reduced cell wall regeneration
(Pearce and Cocking, 1973). Another consideration is that the agarose combined with
higher concentration of sugar used even at the early culture did not play a favorable role
on the protoplasts growth from the beginning culture, but the agarose combined with low
concentration of sugar increase plating efﬁciency and colony formation. The agarose
may increase protoplast stability by immobilizing protoplasts in agarose so that low
osmotic condition of bead culture can be used immediately after plating without bursting
protoplasts. Schnabl and Youngman (1985) reported that immobilization of protoplasts
in alginate leads to an increased stability mainly of a mechanical nature, thus prolonging
the life of the cells. A further possibility is that immobilization may tend to aid cell wall

regeneration of the protoplasts.

32
Effects of humane concentrations of liquid media on plating efficiency and colony

formation in head culture The plating efﬁciency and colony formation in A8 liquid
medium were signiﬁcantly higher than those in A7 and A9 liquid media (Table 1.3).
Hormone concentrations lower or higher than that of A8, signiﬁcantly decreased plating

efﬁciency and colony formation.

Effects of types of culture on plating efficiency and colony formation The ﬁrst cell
division occurred 1 day after plating in bead culture and after 1 week in agarose layer
and liquid cultures. Cells usually divided 1 week after plating in the bead culture and
3 weeks after plating in the other two culture types. The ﬁrst colony formation appeared
3 days after plating in bead culture and after 3 weeks in the other two cultures. Colonies
grew up to 0.48 mm diam. 1 week after plating in bead culture and 0.47 mm diam. after
4 weeks in the other two cultures. Cell division in bead culture was much more rapid
than in the other two cultures.

Bead culture using solid KM6 and liquid A8 media led to a statistically signiﬁcant
increase in plating efﬁciency (19.1% vs. 1.8% in agarose layer culture and 1.3% in
liquid culture) and colony formation (15.5% vs. 2.5% in agarose layer culture and 2.0%
in liquid culture) (Table 1.4). There was no signiﬁcant difference between agarose layer
and liquid cultures for either plating efﬁciency or colony formation. In these
experiments, the same callus-inducing medium, protoplast isolation procedure, and KM
basal medium as those of Elmer et al. (1989) were used except different protoplast
culture methods and cultivars or NAA for asparagus protoplast culture. The plating

efﬁciency (19.1 %) and colony formation (15.5 %) counted 1 week from head culture was

33

Table 1.3. Effects of hormone concentrations of liquid media on plating efﬁciency and

colony formation in bead culture.

 

 

Liquid medium Plating efﬁciency (%)l Colony formation ( %)1
(cell divided / total (colonies > 3 cells /
protoplasts plated) total protoplasts plated)

A 8 19.12 A 15.52 A

A 7 11.53 B 7.54 B

A 9 6.92 C 5.13 C

 

1Means of 6 replicate plates from all three isolations. Means within a column with
different letters are signiﬁeantly different (Duncan’s multiple range test, P < 0.05).

Plating efﬁciency and colony formation was evaluated 1 week after isolation.

34
Table 1.4. Effects of liquid, agarose layer and bead type cultures on plating efﬁciency

and colony formation.

 

 

Type of culture Plating efﬁciency (%)1 Colony formation (%)l
(cells divided / total (colonies > 3 cells /
protoplasts plated) total protoplasts plated)

Bead 19.12 A 15.52 A

Agarose layer 1.75 B 2.48 B

Liquid 1.31 B 1.96 B

 

1Means of 6 replicate plates from all three isolations. Plating efﬁciency and colony
formation were determined 1 week after isolation for the bead culture; plating efﬁciency
was evaluated 3 weeks and colony formation recorded 5 week after isolation for the
agarose layer and liquid cultures. Means within a column with different letters are

signiﬁcantly different (Duncan’s multiple range test, P < 0.05).

35

a signiﬁcant improvement over previous asparagus protoplast regeneration procedures
(Bui Dang Ha et al., 1975; Elmer et al., 1989) in which Elmer et al. reported a
maximum plating efﬁciency of 7.3% obtained 3 weeks after plating. Kong et a1. (1988)
reported that a maximum about 10% of the cells were divided 20 days after plating using
agarose on polypropylene membrane, and sizable colonies formed about 40 days after
growing colonies on the membrane in asparagus protoplast culture.

Bead culture was very effective in promoting protoplast division in this study,
similar to observations made by others (Shillito et al., 1983; Thompson et al., 1986;
Furze et al., 1987). The bead culture technique combines the advantages of high and
low density culture. The protoplasts can be plated close together to maximize
conditioning effects, while a large pool of medium is available for continued growth.
Moreover, the large volume of liquid culture medium dilutes substances released by the

developing cells that may be inhibitory or toxic to the protoplasts (Shillito et al. , 1983).

Comparison of different media on shoot induction Microcalli grew from 0.5 - 1.0 mm
diam. initial to as large as 6 mm diam. within 1 week after transfer from bead culture
to shoot-inducing media (Figure 1.2b). Shoot primordia (light yellow structures, about
1 to 3 mm diam. and 3 mm to 1 cm long) were observed on calli cultured in shoot-
inducing media only with 0.25 mg 1-1 kinetin and 0.25 mg 1-1 or 1 mg 1-1 6-BAP. Four
weeks later, calli were transferred into hormone free MS medium. Green shoot
primordia (1 to 5 mm diam. and 3 mm to 1 cm long) were found on calli cultured on all
shoot-inducing media and the control medium following transferral to hormone-free

medium. Shoots developed within 1 week to 3 months on the hormone-free medium only

36
after transferral from the shoot-inducing media (Figure 1.20). Shoot growth did not

occur on calli cultured on control medium or on calli maintained permanently on media
containing hormones. These results showed that the hormones used in shoot-inducing
media were necessary for induction of the shoots, but inhibited further shoot
development.

The most effetive shoot-inducing medium contained 0.25 mg 1-1 6-BAP combined
with 0.125 mg 1'1 each of NAA and 2,4-D and resulted in an average shoot regeneration
of 92.3% (Figure 1.3). The combination of 6-BAP and auxins was also the most
effective in stimulating the regeneration of shoots from the calli (Figure 1.3). Shoot
formation was not observed after calli were transferred from media with 1 mg 1'1 kinetin
combined with 0.25 mg 1'1 each of NAA and 2,4-D or 1 mg 1'1 ldnetin without auxins
and 0.25 mg 1'1 zeatin and 0.125 mg 1'1 each of NAA and 2,4—D.

Shoots developed directly into plants after calli were transferred from shoot-inducing
media onto hormone-free medium (Figure 1.2d). The plant regeneration was 14% , 16% ,
38% and 79% of total shoots obtained from each shoot-inducing medium respectively
with 0.25 mg-1 6-BAP + 0.125 mg-1 NAA + 0.125 mg-1 2,4-D; 0.25 mg-1 kinetin +
0.125 mg'1 NAA + 0.125 mg’1 2,4-D; 1 mg‘1 6-BAP + 0.25 mg‘1 NAA + 0.25 mg'1
2,4-D and 1 mg'1 6-BAP. After plants were transferred into the greenhouse, 71% of

them survived (Figure 1.4).

CONCLUSION
Critical factors for effective protoplast regeneration were protoplast culture medium,

types of culture, osmotic condition and hormone concentrations of liquid media in bead

37

 

Figure 1.3. Effect of cytokinin type and concentration, in combination with auxins in
the shoot-inducing medium of protoplast-derived calli of asparagus, on shoot regeneration
(%). Level 1: 0.25 mg r1 cytokinin with 0.125 mg 1'1 each of NAA and 2,4-1). Level
2: 1 mg 1‘1 cytokinin with 0.25 mg 1'1 each of NAA and 2,4-D. Level 3: 1 mg l'1
cytokinin alone. Data represent the mean of 3 replicate experiments.

38

 

Figure 1.4. Asparagus plants regenerated from protoplasts growing in the greenhouse.

39

culture and shoot-inducing medium. The high plating efﬁciency, colony formation and
plant regeneration achieved in this investigation offers an opportunity to develop
Fusarium resistant asparagus cultivar through selection of resistant somaclonal variation,

genetic transformation and protoplast fusion.

Literature cited

Bajaj,Y.P.S. 1990. Somaclonal variation- origin, induction, cyropreservation, and
implications in plant breeding, pp. 3—49. In: Biotechnology in agriculture and forestry
11: somaclonal variation in crop‘ improvement I. Y.P.S. Bajaj (ed.). Springer-Verlag
Berlin Heidelberg, Germany.

Bui Dang Ha,D. and Mackenzie,I.A. 1973. The division of protoplasts from Asparagus
oﬁicinalis L. and their growth and differentiation. Protoplasma 78:215-221.

Bui Dang Ha,D. , Norreel,B. and Masset,A. 1975. Regeneration of Asparagus oﬁ‘icinalis
L. through callus cultures derived from protoplasts. J. Exp. Bot. 26:263-270.

Chin,C.K., Kong,Y. and Pedersen,H. 1988. Culture of droplets containing asparagus
cells and protoplasts on polypropylene membrane. Plant Cell Tissue Organ Cult. 15 :59-
65.

Cocking,E.C. and Peberdy,J.F. 1974. The use of protoplasts from fungi and higher
plants as genetic systems. A practical Handbook, University of Nottingham.

Cohen,S.I. and Heald,F.D. 1941. A wilt and root rot of asparagus caused by Fusarium
oxysporum (Schlecht.). Plant Dis. Rep. 25:503-509.

Desjardins,Y. 1992. Micropropagation of asparagus (Asparagus oﬁ‘icinalis L.), pp. 26-
41. In: Biotechnology in agriculture and forestry 19: high-tech and micropropagation III.
Y.P.S. Bajaj (ed.). Springer-Verlag Berlin Heidelberg, Germany.

Elmer,W.H., Ball,T., Volokita,M., Stephens,C.T. and Sink,K.C. 1989. Plant
regeneration from callus-derived protoplasts of asparagus. J. Amer. Soc. Hort. Sci.
114:1019-1024.

Endo,R.M. and Burkholder,E.C. 1971. The association of Fusarium moniliforme with
the crown rot complex of asparagus. Phytopathology 61:891 (Abstr.).

Evans,D.A. and Bravo,J.E. 1983. Protoplast isolation and culture, 1:124-176. In:
Handbook of plant cell culture. D.A. Evans, W.R. Sharp, P.V. Ammirato, Y Yamada
(eds.). Macmillan, New York.

Furze,J.M., Rhodes,M.J.C. and Robins,R.J. 1987. The use of agarose bead culture for
the regeneration of single cell-derived colonies from protoplasts isolated from suspension

40

41
culture of Humulus Iupulus. Plant Cell Tissue Organ Cult. 8: 17-25.

Graham,K.M. 1955. Seedling blight, a fusarial disease of asparagus. Can. J. Bot.
33:374-400.

Grogan,R.G. and Kimble,K.A. 1959. The association of Fusarium wilt with the
asparagus decline and replant problem in California. Phytopathology 49:122-125.

Kong,Y. and Chin,C.K. 1988. Culture of asparagus prot0p1asts on porous polypropylene
membrane. Plant Cell Rep. 7:67-69.

Lacy,M.L. 1979. Effects of chemicals on stand establishment and yields of asparagus.
Plant Dis. Rep. 63:612-616.

Lal,R. and Ial,S. 1990. Protoplasts in crop improvement, pp. 117-137. In: Crop
improvement utilizing biotechnology. R. La] and S. Ial (eds.). CRC Press, Inc. USA.

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

Pearce,R.S. and Cocking,E.C. 1973'. Behavior in culture of isolated protoplasts from
"Paul’s Scarlet Rose" suspension culture cells. Protoplasma 77: 165-180.

Reusink,A.W. 1978. Leucine uptake and incorporation by Convolvulus tissue culture
cells and protoplasts under severe osmotic stress. Physiol. Plant. 44:48-56.

Schnabl,H. and Youngman,R.J. 1985. Immobilization of plant cell protoplasts inhibits
enzyrrric lipid peroxidation. Plant Sci. 40:65-69.

Shillito,R.D., Paszkowski,J. and Potrykus,l. 1983. Agarose plating and a bead type
culture technique enable and stimulate development of protoplast-derived colonies in a
number of plant species. Plant Cell Rep. 2:244-247.

Stephens,C.T., De Vries,R.M. and Sink,K.C. 1989. Evaluation of Asparagus species for
resistance to Fusarium oxyspomm f. sp. asparagi and F. monilifonne. HortScience
24:365-368.

Stephens,C.T., Kusnier III,J.J. and Hausbeck,M.K. 1991. Control of asparagus root rot
using root dips and foliar sprays, 1990. Fungicide and Nematicide Tests.

Stephens,C.T. and Sink,K.C. 1992. Asparagus decline syndrome and replant problem
in Michigan, pp. 23-24. Department of Botany and Plant Pathology and Horticulture,
Michigan State University.

Thompson,J.A., Abdullah,R. and Cocking,E.C. 1986. Protoplast culture of rice (Otyza

42
sativa L.) using media solidiﬁed with agarose. Plant Sci. 47: 123-133.

Van Bakel,J.M.M. and Kerstens,].J.A. 1970. Root rot in asparagus caused by Fusarium
oxysporum f. sp. asparagi. Neth. J. Plant Pathol. 76:320-325.

CHAPTERII

The Development of Asparagus oﬂiciualis cv. Lucullus 234 Somaclones with High

levels of Resistance to Fusarium spp.

ABSTRACT

Asparagus somaclones were produced by subculturing shoots initially generated from
callus—derived protoplasts of A. oﬂicinalis cv. Lucullus 234 through callus cycles for over
a three year period. One hundred and twenty protoplast-derived somaclones of ’Lucullus
234’ were screened for resistance to two virulent Michigan isolates each of Fusarium
oxysporum f. sp. asparagi Cohen and Heald (FOA 10 and FOA50) and F. proliferatum
(T. Matsushima) Nirenberg (FM12 and FM49) in the greenhouse. Somaclones had
signiﬁeantly increased levels of resistance overall to both Fusarium spp. over the
vegetatively micropropagated ’Lucullus 234’ parental plants. After somaclones
containing higher levels of resistance (at rating scale 1 or 2) were vegetatively
nricropropagated, a minimum of 12 micropropagated plants of each were rescreened for
resistance to the most virulent isolate FOA50 in the greenhouse. Two somaclonal lines,
R7 and R4, were rated as highly signiﬁcantly resistant to FOA50 in comparison with
the vegetatively micropropagated parental plants. These lines were very similar

horticulturally to the parental cultivar. Cytogenetic studies showed that R7 and R4 were

43

44
diploid with normal chromosomal number (2n =20) as ’Lucullus 234’ parental cultivar

(2n =20). Genomic ﬁngerprints by randomly ampliﬁed polymorphic DN As (RAPDs)
showed that the resistant somaclonal lines R7 and R4 may have carried DNA sequence

mutations as their DNA sequences varied from the ’Lucullus 234’ parental plant.

INTRODUCTION

Since the ﬁrst successful methods for in vitro growth of carrot (Gautheret, 1939;
Nobecourt, 1939) and tobacco (White, 1939) tissues were reported in 1939, tissue-culture
techniques had been subsequently developed and reﬁned for hundreds of plant species.
Two observations noted during preliminary studies of cultured cells and regenerated
plants in vitro are that: 1) genetic and cytogenetic variation commonly increases during
exposure of cells to in vitro growth, and 2) while much phenomenology has been
recorded, the mechanisms generating the variation remain to be described. Early variant
plants regenerated from cell cultures of geranium were termed ”calli-clones” by Skirvin
and J anick (1976a), while plants regenerated from protoplasts of potato were termed
"protoclones' by Shepard et al. (1980). It was ﬁnally in 1981 that Larkin and Scowcroft
named the genetic variability in plants regenerated from tissue and cell culture as
”somaclonal variation" , a term which has been widely accepted (Iarkin and Scowcroft,
1981).

Somaclonal variation comes from: 1) preexisting differences in individual somatic
cells revealed by enabling these to regenerate in differentiated plants by tissue culture and
2) culture-induced changes (Bajaj, 1990). Somaclonal variation has resulted in epigenetic

variation and genetic variation. Epigenetic changes are usually induced by tissue culture

45

condition and are not expressed in the sexual progeny of regenerated plants. Genetic
changes have been detected either in spontaneously generated mutants or as a result of
induced mutations (Evans, 1989). A earlier major achievement in this direction was
made by Skirvin and J anick (1976a), although their work did not receive much attention
at that time. They recovered variant plants differing in their oil component, fasicination,
pubescence, and anthocyarrin production from callus cultures of scented geranium.
Extensive studies conducted during the last decade have shown that the cell cultures,
especially in long term cell cultures, undergo various morphological and genetic changes
including polyploidy, aneuploidy, chromosome breakage, deletion, translocation, gene
ampliﬁcation, inversion, single gene mutation, etc. (Meins, 1983; D’Amato, 1985). In
the early 19803, Pelletier and Pelletier (1971) had found such culture-induced variations
including chlorophyll deﬁciency and enhanced axillary shooting in lettuce cultures.
Interestingly, in all plants which showed such variations the chromosomal number was
normal. Further genetic-based information from anther culture-induced variations only
recognized ploidy changes and chromosomal number responsible for such variations
(Devreux and Ianeri, 1974; Collin and Legg, 1980; Oono, 1981). However, Alhoowalia
(1976 and 197 8) for the ﬁrst time reported gross structural changes including reciprocal,
translocation, deletion and inversions responsible for the production of variants in
Lolium. In addition, the molecular basis of somaclonal variation has been studied. The
detected change of somaclonal variant in maize was a single base pair alteration that
resulted in a change from glutamic acid to valine in the last codon of exon 6 (Brettell et
al. , 1986). Nuclear ribosomal DNA spacer length variation has been found in

somaclonal variants of a spring wheat (Rode et al. , 1987). Grossly altered methylation

46

pattern, which resulted in epigenetic variation, have been reported in somaclonal variants
(Brown and Lorz, 1986). Qualitative and quantitative changes in gliadin proteins have
been detected in somaclonal variants of wheat (Maddock et al. , 1985; Cooper at al. ,
1986). Organelle DNA changes were found in somaclonal variants of potato (Kemble
and Shepard, 1984).

The ﬁrst work recognizing the potential of somaclonal variation as a source of
variability for crop improvement was conducted with sugareane (Heinz and Mee, 1969)
and later with potatoes (Heinz, 1973; Nickell, 1977; Shepard et al., 1980). These
workers found that plants regenerated from callus and protoplast cultures varied in
morphological characteristics, maturity date, yield, and response to pathogens. Since
then tremendous progress in this area have been made. Agriculturally useful sorrraclonal
variants have been identiﬁed which have desirable traits such as increased solids in
tomato, male sterility in tomato and rice, higher yields and enhanced protein production
in rice, earliness in maize, freezing tolerance in wheat, and increased sugar content in
sugarcane (Evans, 1989; Bajaj, 1990). Leaf, ﬂower and color variants have been used
to develop new breeding lines of omamentals (Evans and Bravo, 1986).

Development of resistance to fungal, bacterial and viral diseases in various crops has
been the major contribution of somaclonal variation (lal and Lal, 1990). In alfalfa,
somaclones resistant to Fusarium oxysporum were regenerated from cell lines selected
for resistance to Fusarium culture ﬁltrates (Hartman er al. , 1984). Screening of alfalfa
somaclones regenerated from protoplasts resulted in plants that were resistant to
Verticillium albotrum (Iatunde and Lucas, 1983). Two somaclonal lines of rice were

resistant to sheath blight in the ﬁeld (rccp, 1987). Daub (1986) has reported

47

protoplast-derived somaclones of tobacco cultivars with increased resistance to several
major tobacco pathogens including TMV, Meloidogyne incognito, and Phytophthora
parasitica var. nicotianae. She also identiﬁed three somaclones whose progeny enhanced
resistance to black shank in both ﬁeld and greenhouse. Resistance in celery to Fusarium
oxysporum f. sp. apil' race 2 has been enhanced with somaclonal variation (Toth and
lacy, 1991). Somaclonally-derived resistance to tomato mosaic virus and tobacco
mosaic virus has been obtained in tomato (Smith and Murakishi, 1993). To date, six
cultivars have been released that were derived from somaclonal variation. The sugarcane
cv. Ono, which is higher yielding and shows increased resistance to Fiji disease, was
developed by isolating subclones of sugarcane resistant to Fiji disease from a susceptible
cultivar (Kirshnamurthi and Tlaskal, 1974). Velvet Rose, a scented geranium cultivar,
has been developed by Skirvin and Janick (1976b). The sweet potato cultivar Scarlet,
isolated from meristem tip culture-derived clones, has a desired quality trait of darker
and more stable skin color (Moyer and Collins, 1983). DNAP 9 and DNAP 17, two
tomato cultivars, have been shown to have higher solids in several ﬁeld trails and
resistance to Fusarium Race 2, respectively. The two cultivars were derived from plants
regenerated from leaf explants (Evans, 1987). Bell sweet, a yellow bell pepper cultivar,
was identiﬁed as a variant among plants regenerated from anther culture and was shown
to have few or no seeds (Morrison and Evans, 1988).

To identify possible sources of resistance to Fusarium oxysporum and F.
proliferation, ninety ﬁve cultivars and breeding lines of four asparagus species,
Asparagus oﬂ‘lcinalis, A. denslﬂorus, A. setaceus and A. acutlfolius, were evaluated in

response to one virulent Michigan isolate each of F. oxysporum (FOAlO) and F.

48
proliferation (FM12) in the greenhouse tests. A cultivar of A. oﬁ'icinalis, Lucullus 234,

had the highest resistance to FOA10 and FM12 among 90 cultivars and breeding lines
of this species tested. Two cultivars of an ornamental asparagus species, A. denslﬂorus
’Sprengeri’ and ’Myersii’ were more resistant than any other asparagus cultivar or
species tested (Stephens et al. , 1989). However, sexual crosses of A. oﬁ‘icinalis with the
resistant species, A. densiﬂorus, have been unsuccessful, probably due to incompatibility
barriers (Elmer et al. , 1989).

A previous study by Dr. Smither in this laboratory screened somaclones of 2 cultivar
of A. oﬁ'icinalis, ’Lucullus 234’ and ’Jersey Giant’. Somaclones of ’Lucullus 234’
exhibited a higher level of resistance to F. oxysporum and F. proliferation than the
’Jersey Giant’ somaclones. In addition, somaclones of ’Lucullus 234’ were more
resistant to F. proliferation than F. oxysporum (M.L. Smither and C.T. Stephens,
unpublished observation).

The genome provides a source of constant primary character, and the cultivar
identiﬁcation, measuring levels of genetic diversity, genetic mapping, tagging genes of
interest in many cultivated plants have been done by using randomly ampliﬁed
polymorphic DNAs (RAPDs) analysis (O’Brien, 1990; Quiros et al. , 1991). Although
the genetic bases of somaclonal variations in other crops have been determined by the
techniques described above, interest in RAPDs is rapidly growing because their
identiﬁcation is simple and fast. In addition, their polymorphisms among the
ampliﬁcation products are detected frequently and through examination of an ethidium
bromide-stained agarose gel instead of using radioactivity (Williams et al. , 1990). The

RAPD PCR technique has been developed by Williams et al. (1990). This technique

49

relies on the differential enzymatic ampliﬁeation of random DNA fragments using PCR
with single primers of arbitrary nucleotide sequence (usually lO-mers), but require no
knowledge of target DNA sequence. Polymorphisms result from either chromosomal
changes in the ampliﬁed regions or base changes that alter primer binding. The RAPD
markers are usually dominant beeause polymorphisms are detected as the presence or
absence of bands. RAPD markers can be used for genetic mapping, identiﬁcation of the
genomic regions derived from the trait donor parent of near-isogenic lines and F2 pool
selection, study of population genetics and molecular taxonomy (Williams et al. , 1993).
The RAPD markers linked to disease resistant genes have been successfully identiﬁed in
tomato (Martin et al., 1991) and lettuce (Michelmore et al., 1991). The RAPD PCR
was also used to identify grapevine cultivars (Collins and Symons, 1993).

The goal of this study was to select protoplast-derived somaclones of ’Lucullus 234’
with higher levels of resistance to two Michigan virulent isolates each of Fusarium
oxyspomm (FOA10 and FOA50) and F. proliferation (FM12 and FM49) in the
greenhouse, test the stability of the resistance of selected individual more resistant
somaclones, and ﬁnally characterize the genetic basis of the resistant somaclones. In this
investigation, we report: 1) development of a protocol for the production of protoplast-
derived somaclones , 2) development of a protocol for vegetative micropropagation of
’Lucullus 234’ seed plants, 3) the screening of 120 protoplast-derived somaclones of
’Lucullus 234’ for resistance to FOA10, FOA50, FM 12 and FM49 in the greenhouse,
4) the rescreening of a minimum of 12 vegetatively nricropropagated plants of each more
resistant somaclone in response to FOA50 in the greenhouse, 5) a preliminary

characterization of the genetic basis of the resistant somaclones using the RAPD PCR and

50

chromosome counts.

MATERIALS AND METHODS

Production of somaclones Due to the higher Fusarium resistance of ’Lucullus 234’
previously reported (Stephens et al. , 1989) and the highest probability to get variants
from plants regenerated from protoplasts compared with other types of culture such as
eallus, stems, somatic embryos, adventitious buds and etc. (Demarly, 1986), initial
shoots regenerated from callus-derived protoplasts of A. oﬁ'icinalis cv. Lucullus 234 (Dan
and Stephens, 1991) were used to produce somaclones. These shoots were cultured on
Murashige & Skoog (MS) medium (1962) supplemented with 0.1 mg/l 6-BAP (RM 17),
calli formed on the shoots within 15 to 30 days, and new shoots developed on the calli
within 30 days. By using this technique, shoot production was maintained for over 3
years and the produced shoots were used as the somaclonal source because accumulation
of variability has been positively correlated with duration of culture (Evans et al. , 1984).
Effects of different root inducing agents: auxins (NAA, 2,4-D, IAA and IBA), ancymidol
and sucrose at different concentrations and combinations on root differentiation of the
produced shoots were exanrined. Rooted plants were cultured in a hormone-free MS
medium for shoot and root elongation for another month. All cultures were placed at
27° c with 40 uEm'zs'l light and a 16 h photoperiod.

Before plants were transferred to the greenhouse, they were acclimatized in order to
adapt to greenhouse conditions. Plants were transferred to pots containing synthetic

media (Baccto, Michigan Peat Co. , Houston, Texas) (1 vermiculite : 1 Peat : l Perlite).

51
The pots were placed in a plastic bag at 22 to 24° C under cool-white ﬂuorescent light

and a 24 hour photoperiod. The bags were completely sealed for the ﬁrst week, 3 holes
punctured in the sealed bag for the second week, and opened for the third week. The
pots were maintained one additional week after the plastic bag was completely removed

at the end of the third week, and then were moved to the greenhouse.

Vegetative micropropagation of ’Lucullus 234’ Due to limited number of ’Lucullus
234’ seeds, it was necessary to develop a protocol to vegetatively micropropagate
’Lucullus 234’ plants. Two different concentrations of cytokinin 6-BAP were tested for
shoot production. Spears of greenhouse grown ’Lucullus 234’ were surface sterilized
(Chapter I) and cut into single-node segments. Two to four segments were cultured on
20 nrl of either one of two different modiﬁed MS media supplemented with 0.5 mg/l
(RM18) and 0.1 mg/l (RM17) of BAP in 25 X 150 mm culture tubes. The effects of
four different auxins 1AA, IBA, NAA, and 2,4-D on root induction were tested.
Branched shoots at the node region were excised after 15 day culture in RM17 medium.
Two shoots were cultured in a culture tube having 20 ml each of four different root-
inducing media containing basic MS medium, and 1AA, IBA, NAA, and 2,4-D,
respectively, each at concentration of 2 mg/l. IBA was found to result in the highest root
production. Further, ﬁve concentrations of IBA, 3 mg/l, 5 mg/l, 7 mg/l, 9 mg/l and 11
mg/l, were tested for improving rooting efﬁciency. Rooted plants were cultured on a
hormone-free MS medium for further shoot and root development for one month. All
cultures were maintained at 27° C with 40 rtEm'zs'l light and a 16 h photoperiod. Plants

were transferred into the greenhouse after acclimazation as described above. All

52

experiments contained at least 8 replicates per treatment and were repeated 4 times.

The first cycle of screening for higher levels of Fusarium resistance in somaclones
in the greenhouse One hundred and twenty somaclones and 120 vegetatively
nricropropagated plants of ’Lucullus 234’ generated as described above were used for
screening to the Fusarium isolates. Previous work demonstrated that a cultivar of A.
oﬁ‘icinalis, ’UC 157’, is highly susceptible to F. oxysporum and F. proliferatum, and a
cultivar of an ornamental asparagus species A. sprengeri, ’Sprengeri’, is highly resistant
(Stephens et al. , 1989). ’UC 157’ and ’Sprengeri’ were used as susceptible and resistant
controls, respectively, for the screening test.

Seeds of Asparagus spp. were surface disinfected according to the method of
Stephens et al. (1988), and germinated in agar medium (1.5%, w/v) at room
temperature. One week later, germinated seeds were transferred into pots containing
synthetic medium as described above and grown in the greenhouse. Inocula of two
Michigan virulent isolates each of F. oxyspomm (FOA10 and FOA50) and F.
proliﬁrratum (FM12 and FM49) were prepared using the method of Wacker et al. (1990).
Each Fusarium inoculated and Fusarium-free soils (control soils) were amended with soil
mix. One-month-old seedlings of ’UC 157’ and ’Sprengeri’, and one-month-old-
greenhouse grown somaclones and vegetatively micropropagated plants of ’Lucullus 234’
were transferred to 53 x 28 x 6 cm tray containing 7 kg of Fusarium infested soil.
Plants were fertilized (Peters 20N-20P-20K, 200 ppm) once per week.

Plants were harvested after 1 to 4 months depending on weather conditions such as

temperature or humility. Disease incidence was assessed using a visual rating scale

53
(Stephens et al. , 1989; Wacker et al. , 1990): l= healthy plant, no evidence of disease

(highly resistant), 2= few root lesions, (25% diseased (resistant), 3= moderate number
of root lesions, <50% (moderately susceptible), 4= many root lesions, <75%
(susceptible), and 5 = all roots ﬂaccid or dead (highly susceptible). Experiments were
designed as a two factorial experiment using randomized complete block design for
Fusarium spp. as factor A and germplasm entries as factor B , which is a split plot on
A. All experiments were arranged on greenhouse benches under a natural photoperiod

at 25 to 30°C. Data were analyzed by MSTAT program for ANOVA.

Test stability of the Fusarium resistance in the more resistant somaclones selected
from the first cycle To deternrine if Fhsarium resistance in the individual somaclones
with higher levels of resistance selected from the ﬁrst cycle remain stable, somaclones
with a disease rating of 1 and 2 were maintained in the greenhouse for further disease
screening. Each of the selected somaclones was vegetatively micropropagated using the
method described above except RTM30 medium was used for root induction. At least
twelve rrricropropagated plants of each of the more resistant somaclones were rescreened
for resistance to FOA50 as described above. FOA50 was chosen as the screening agent
in these tests because FOA50 was found to be more virulent to ’Lucullus 234’ and the
controls. All experiments were conducted as a randomized block design in the
greenhouse under the same conditions as described above. Data were subject to

Student’s test.

54
Cytogenetic study of the resistant somaclones In order to examine changes of ploidy

of the somaclones with increased resistance (R4 and R7), their root tips were treated in
0.2 % l-bromonaphthaline for 3 hours and ﬁxed in acetic acid: alcohol (1 :3) for 24 hours
after rinsing in running tap water. They were rinsed in distilled water, macerated in l
N HCl at 60°C for 15 min, and stained in acetocarmine (1%, w/v) for 24 hours then
squashed for observation of chromosomes under a compound microscope. Pictures of
chromosome counts were taken under a laser scanning confocal nricroscope (LSM) Model
210 (Carl Zeiss, Inc. , Thomwood, N .Y.) with a Matrix Multicolor computerized camera

unit (Agfa—Matrix, Orangeburg, N .Y.) which receives signals directly from the LSM.

Determining the genetic bases of the resistant somaclones by RAPDs The more
resistant somaclonal lines R7 and R4 selected from the second cycle of screening and
four randomly chosen seed plants of ’Lucullus 234’ (controls) were screened for RAPDs
by using 20 arbitrary primers to preliminarily measure genetic variability in the resistant
somaclones.

Genomic DNA was isolated from shoots of greenhouse grown plants using the
protocol of Dellaporta et al. (1985). The isolated DNA was then puriﬁed according to
the method of Fang et al. (1992) to remove polysaccharides which inhibit the activity of
many molecular biological enzymes, such as restriction enzymes and Taq polymerase
(Fang et al. , 1992). Two separate DNA isolations were made, to document the
reproducibility of the ampliﬁcation products.

PCR was carried out with a 110S Programmable Coy Temprcler H (COY

corporation, Gass lake MI), 20 arbitrary primers (Table 2.1) (Operon Technologies,

Table 2.1. Source of twenty primers used to screen the resistant somaclones R7 , R4 and

four control plants.

55

 

 

 

Primer [ Sequence Primer Sequence

A CCTACACGGT K ACTGGGCCTC
B CAGCCGAGAA L GACGCAGC'I'I‘
C GAAGGAGGCA M CCGAGGTGAC
D ’ITGCGGCTGA N GGTGCGCACT
E CCCGATCAGA O CACGAACCTC
F CCGCAGTCTG P TCCCGGTGAG
G GGAAAGCGTC Q TGAACCGAGG
H CTCTGCCTGA R GTGTCCCTCT
I CCCCTCAGAA S GGACAAGCAG
J GGTTGGAGAC T CTCCGCACAG

 

56
Alameda, CA) and GeneAmp PCR Core Reagents (Perkin Elmer Cetus, Norwalk CT).

To determine the optimal concentration of MgClz for PCR of asparagus genomic DNA,
1.5 to 5 mM of MgC12 at 0.5 mM interval was used. The concentration of 1.5 mM of
MgC12 was chosen for PCR because it was found to be the only concentration which
provided PCR products. PCR reaction mixtures contained 25 p1 of IX PCR buffer (500
mM KCl, 100 mM Tris-HCl, pH 8.3), 200 pM each of dATP, dCTP, dGTP, and dTI‘P,
0.4 pM primer, 33 ng/pl T4 gene 32 protein (Pharmacia Biotech Inc. Piscataway NJ),
0.04 will AmpliTaq DNA polymerase, 1.5 mM MgC12 and 2 ng/pl genomic DNA, and
the reaction mixture overlaid with 25 pl of sterile mineral oil (Sigma Chemical Co. , St.
Louis, MO). The PCR program consisted of an initial denaturation of 7 min at 94°C,
45 cycles of 1 min at 94°C, 1 min at 35°C, 2 min at 72°C, and a ﬁnal extension step
of 15 min at 72°C. The PCR products were size-separated by electrophoresis in 1.4%
agarose (Sigma Chemical Co., St. Louis, MO) in 1X TAE at 2.5 V/cm in a cold room

(4°C) for 24 hours.

RESULTS AND DISCUSSION

Production of somaclones Initial shoots generated from callus-derived prot0p1asts of
A. oﬁ‘icinalis (Dan and Stephens, 1991) were cultured on shoot-inducing medium RM17.
Fifteen to 30 days later, calli were produced on the shoots, and new shoots developed
on the ealli within 30 days. Shoot production was maintained for over 3 years using this
procedure. When the produced shoots were transferred into different root-inducing

media, shoots formed a small compact mass resembling a crown where the thick roots

57

were attached, and then roots produced from the mass. The highest average root
production (55.5%) was obtained from shoots cultured four weeks in a MS medium
containing IBA at 11 mg 1'1 (data not shown). Further root development was much
quicker when shoots with induced roots were cultured in a hormone-free MS medium as
opposed to shoots cultured in initial root-induction media. This indicated that IBA,
which is necessary for root induction, inhibited further root growth. Rooting of shoots
regenerated from protoplasts in this study was not improved by ancynridol or by
increasing the concentrations of sucrose in contrast to the results reported by Chin (1982)
and Desjardins et al. (1987) (data not shown). Two hundred plants were transferred into

the greenhouse, 80.5 % of them survived and were used as a source of somaclones.

Vegetative micropropagation of ’Lucullus 234’ Shoots (1 to 2 cm in length) developed
from more than 90% of nodes of spears 15 days after single-node segments were cultured
on RM17 medium. Roots appeared usually 15 to 30 days after the produced shoots were
cultured in root-inducing media. Among four different auxins tested, IBA produced the
highest rooting of 33.3% in one month (Figure 2.1). The auxin, 2,4-D, completely
inhibited root induction (Figure 2.1). The highest average of 57.5% of shoots having
roots was obtained 1 month after the shoots were cultured in RTM 26 containing MS
medium + IBA 3 mg/l (Figure 2.2). One month after culture in RTM26, all the
plantlets possessed well-developed crowns with 4 to 7 spears and vigorous roots (Figure
2.3). At this stage, plants were transferred to a hormone-free MS medium for further
shoot and root development for one month. Two hundred and ﬁfty seven plants were

transferred to the greenhouse after acclimazation as described above, and 84.4% of them

58

Rooting Frequency (%)
40

33.3

30

20

10

 

Types of Auxin
E IBA (2 mgll) lllll NAA (2mg/I) IAA (2 mgll) I 2.4-D (2 mgll)
Figure 2.1. Effects of different auxins (IAA, IBA, NAA, and 2,4-D) on rooting

frequency (%) of the shoots produced on RM17 medium of asparagus cv. Lucullus 234
after 4 weeks in culture.

59

Rooting Frequency (%)

 

 

_._________________________________________________A.

.77777777777777777

 

 

Eng/I llllegll Smgll E7mg/l 9mg/II11mgll

rooting frequency (%) of the shoots
. Lucullus 234 after 4 weeks in culture.

Effects of [BA concentrations on

produced on RM17 medium of asparagus cv

Figure 2.2.

 

Figure 2.3. Four-week-old plantlets of asparagus cv. Lucullus 234 in RTM26 medium
containing basic MS medium + 3 mg/l IBA.

61

survived.

Screening of somaclones for Fusarium resistance For the ﬁrst cycle of screening
somaclones for Fusarium resistance, 120 somaclones of ’Lucullus 234’, and 120 plants
of vegetatively nricropropagated ’Lucullus 234’ parental plants, ’Sprengeri’ and ’UC 157’
were screened for resistance to FOA10, FOA50, FM12 and FM49. Both different
Fusarium spp. and germplasm had a signiﬁcant effect on Fusarium disease incidence
(Table 2.2). The interaction between germplasm and Fusarium spp. did not signiﬁcantly
affect Fusarium disease incidence (Table 2.2). FOA50 was signiﬁcantly more virulent
to all plants when compared to FOA10 (data not shown). Somaclones were signiﬁcantly
more resistant to both Fusarium spp. when compared to the vegetatively micropropagated
’Lucullus 234’ parental plants (Table 2.3). Of 120 somaclones, 24.2% of them (vs.
8.3% of the parental plants) fell into rating scale 1, 40.8% (vs. 41.6% of the parental
plants) in rating scale 2, 15.8% (vs. 25.8% of the parental plants) in rating scale 3, 8.3%
(vs. 10.8% of the parental plants) in rating scale 4, and 10.8% (vs. 13.3% of the
parental plants) in rating scale 5 (Table 2.4). Of 120 plants of ’Sprengeri’, 50.8% of
them fell into rating scale 1, 47.5% in rating scale 2, 1.6% in rating scale 3 and none
in rating scale 4 and 5. Of 120 plants of ’UC 157’, none of them fell into rating scale
1, 17.5% in rating scale 2, 40.8% in rating scale 3, 27.5% in rating scale 4 and 14.2%
in rating scale 5 (Table 2.4). Ninety two percent of the somaclones and vegetatively
micropropagated plants of ’Lucullus 234’, and ’Sprengeri’ remained healthy when they
were planted in control soils amended with soil mix without the Fusarium isolates, and

75% of ’UC 157’ were healthy. The rest of all the plants had a few root lesions (at

62

Table 2.2. Analysis of variance of a randomized complete block design for Fusarium

spp. as factor A and germplasm entries as factor B, which is a split plot on A.

 

 

 

 

 

Source Degrees of Mean F-test"
freedom square

Block 5 0.522 NS

Factor A (Fusarium spp.) 3 1.658 S

Error 15 0.353

Factor B (Germplasm) 3 14.840 S

AB 9 0.533 N S

Error 60 0. 384

Total 95

 

"NS = F-test not signiﬁcant.

S = F-test signiﬁcant (P<0.05).

63

Table 2.3. Mean disease rating of asparagus plants screened with Fusarium oxysporum

(isolates FOA10 and FOA50) and F. proliferaturn (isolates FM12 and FM 49) in the

 

 

 

 

 

greenhouse.
Treatments" 1 Total mean

Plant source disease

. I FM12 l FM49 FOA10 FOA50 | rating’
’UC157’ 3.47 3.1 3.00 3.97 3.38 A
(susceptible control)
Micropropagated 2.77 2.50 2.67 3.23 2.79 B
seed plants of
’Lucullus 234’
Somaclones of 2.23 2.90 1.80 2.70 2.41 C
’Lucullus 234’
’Sprengeri’ 1.63 1.47 1.40 1.53 1.51 D

(resistant control)

 

"One hundred and twenty plants of each plant sources were screened at four different
times. Disease was assessed using a visual rating scale: 1= no disease, 2: < 25%
diseased, 3= < 50% diseased, 4= < 75% diseased, and 5= > 75% diseased.

yMeans disease rating of four treatments (FM12, FM49, FOA10 and FOA50). Means
within a column with different letters are signiﬁcantly different (Duncan’s multiple range

test, P < 0.05).

64

Table 2.4 Disease rating of asparagus plants screened with Fusarium oxysporum

(isolates FOA10 and FOA50) and F. proliferation (isolates FM12 and FM49) in the

 

 

 

 

 

 

 

 

greenhouse.
Plant source No. of plants in disease rating Mean
scales" disease
° y
1 2 I 3 4 5 mung
’UC157’ 0 21 49 33 17 3.38 A
(Susceptible control)
Micropropagated 10 50 31 13 16 2.74 B
seed plants of
’Lucullus 234’
Somaclones of 29 49 19 10 13 2.41 C
’Lucullus 234’
’Sprengeri’ 61 57 2 0 0 1.51 D
(Resistant control)

 

"One hundred and twenty plants of each of plant source were screened at four different

times. Disease was assessed using a visual rating scale: l= no disease, 2= < 25%

diseased, 3= < 50% diseased, 4= <75% diseased, 5= >75% diseased.

yMeans of disease rating within a column with different letters are signiﬁcantly different

(Duncan’s multiple range test, P < 0.05).

65

rating scale 2) when they were planted in the control soil (data not shown).

To determine stability of the Fusarium resistance in the most resistant somaclones
selected from the ﬁrst cycle, 54 somaclones with a disease rating of 2 or less were
maintained in the greenhouse for further disease screening. Of 54 somaclones maintained
in the greenhouse, 15 (27.7%) of them developed spears which could be used as explants
for micropropagation. The 15 somaclones were then vegetatively micropropagated as
described above. Only two somaclones (R7 and R4) could develop healthy roots in vitro.
At least 12 micropropagated plants of each of R7 and R4 were rescreened with FOA50
in the greenhouse using a randomized complete block design. Both somaclonal lines R7
and R4 were highly signiﬁcantly resistant to FOA50 in comparison with the vegetatively
rrricropropagated ’Lucullus 234’ parental plants (Table 2.5 and 2.6). Of 12
micropropagated plants of R7 , 91.7% of them were rated at 1 or 2 in contrast to 6.67%
of the parental plants (Table 2.5). Of 18 micropropagated plants of R4, 50% of them
were rated at 1 or 2 in contrast to 6.7% of the parental plants (Table 2.6). These results
indicate that the increased levels of resistance of R7 and R4 to the Fusarium isolates from
the ﬁrst cycle of screening is stable after vegetative micropropagation. In addition, these
lines were very similar horticulturally to the parental cultivar.

FOA10 had signiﬁcantly lower virulence to all plants tested when compared to
FOA50. Although ’Lucullus 234’ was the most resistant to FOA10 and FM12 in
comparison with 90 cultivars and breeding lines of this species tested (Stephens et al. ,
1989), our results showed that the vegetatively micropropagated plants of ’Lucullus 234’
seed plants were as susceptible to FOA50 as ’UC 157’ (Table 2.5 and 2.6)

Asparagus somaclones of ’Lucullus 234’ expressed a higher level of resistance to

66

Table 2.5. Disease rating of vegetatively rrricropropagated plants of the more resistant

somaclone R7 rescreened with Fusarium oxysponon (isolate F OA50) in the greenhouse.

 

 

 

 

 

Plant source No. of plants in disease rating Mean
scales" disease
5 ratingy
r j 2 | 3 | 4 | 5
’UC157’ 0 1 9 2 3 3.47 A
(susceptible control)

Micropropagated seed plants of 0 1 12 1 1 3.13 A
’Lucullus 234’

Micropropagated plants of 3 8 1 0 0 1.83 B
somaclone R7 of
’Lucullus 234’

’Sprengeri’ l4 1 0 0 0 1.07 C
(resistant control)

"Twelve vegetatively micropropagated plants of R7 were screened with FOA50. Disease
was assessed using a visual rating scale: 1= no disease, 2= < 25% diseased, 3: <
50% diseased, 4= < 75% diseased, and 5= > 75% diseased.

’Means within a column with different letters are signiﬁcantly different (Student’s t test,

P < 0.01).

67

Table 2.6. Disease rating of vegetatively micropropagated plants of the more resistant

somaclone R4 rescreened with Fusarium oxysporum (isolate F OA50) in the greenhouse.

 

 

 

 

 

Plant source No. of plants in disease rating Mean
scales" disease
5 ’ ratingy
l ] 2 l 3 [ 4 [ 5
’UC157’ 0 1 5 5 4 3.80 A
(susceptible control)

Micropropagated seed plants of 0 1 7 4 3 3 . 60 A
’Lucullus 234’

Micropropagated plants 1 8 6 2 1 2.67 B
of Somaclone R4 of

’Lucullus 234’

’Sprengeri’ 14 1 0 0 0 1.07 C

(resistant control)

"Eighteen vegetatively nricropropagated plants of R4 were screened with FOA50.
Disease was assessed using a visual rating scale: 1= no disease, 2= < 25% diseased,
3= < 50% diseased, 4: < 75% diseased, and 5= > 75% diseased.

3’ Means within a column with different letters are signiﬁcantly different (Student’s t test,

P < 0.01).

68

the Fusarium isolates than the vegetatively nricropropagated ’Lucullus 234’ parental
plants after the ﬁrst cycle of screening. The vegetatively micropropagated plants of 2
somaclones with higher resistant levels were rescreened to the most virulent isolate
FOA50. These two somaclonal lines, R7 and R4, were rated as highly signiﬁcantly
resistant in comparison with the parental plants, indicating the maintenance of the
increased resistance in R7 and R4 lines after vegetative micropropagation. Highly
resistant somaclones were regenerated at a high frequency of 24.2 % (vs. 8.3 % in the
parental plants of ’Lucullus 234’) from the moderately resistant cultivar Lucullus 234
after the ﬁrst cycle of screening. Ninety two percent of the vegetatively rrricropropagated
plants of R7 had disease ratings of 1 or 2, whereas 93.3% of the parental plants had
rating of 3 or greater. Fifty percent of the vegetatively rrricropropagated plants of R4

had rating of l or 2, while 93.3% of the parental plants had rating of 3 or more than 3.

Measuring genetic variability in the resistant somaclones In order to understand the
origin of the resistant somaclones, R7 and R4 were ﬁrst cytogenetically studied using
chromosome counts. Both somaclonal lines showed diploid with normal chromosome
numbers (2n = 20) as their ’Lucullus 234’ parental cultivar (2n=20)(Figure 2.4, Figure
2.5 and Figure 2.6). Similar results were reported by Sibi (1976).

To examine for possible DNA sequence variations in the resistant somaclones, RAPD
PCR was carried out. In our initial attempts to optimize RAPD PCR analysis in
asparagus, only one concentration at 1.5 mM of MgClz gave rise to PCR products (data
not shown). Reproducible RAPD products were obtained by using T4 gene 32 protein

(gp32). In the absence of gp32 in the reaction mixture, the yield of products and the

69

 

Figure 2.4. The chromosomes in A. oﬁ‘icinalis cv. Lucullus 234 (2n = 20).

70

°°ef’t

o \\
e‘ 1"}.
.‘h-
t‘ ‘ ,.
'1. '0.’
z... 2”
.,le

I "

e r '. f D o . ’1.“" '
‘C I. . ~ ‘71,. .‘ l
0.. o -‘ . '-.k‘
_ ‘ - 0 a. ‘ ..o- “
e e " - .
0“... e.. I . ‘...' J
2.. ._ e a ‘ . '. ‘ .9: SP“
.. 1’ ') .— ’. M J

 

Figure 2.5. The chromosomes in the resistant somaclone R7 of A. oﬁ‘icinalis cv.
Lucullus 234 (2n = 20).

71

 

ﬁgure 2.6. The chromosomes in the resistant somaclone R4 of A. oﬁ'icinalis cv.
Lucullus 234 (2n = 20).

72

number of bands varied between PCR reactions, especially in the lower molecular weight
range (data not shown). An improvement in the yield and reproducibility of PCR
products by gp32 has also been reported by Schwarz et al. (1990), Panaccio and Lew
(1991) and Collins and Symons (1993).

The resistant somaclonal lines R7 and R4 developed from the second cycle of
screening and four ’Lucullus 234’ seed plants (controls) were screened for RAPDs using
20 arbitrary primers. Of the 20 primers, 75 % them generated unique ampliﬁcation
products in R7, that were not found in the four control plants (Figure 2.7, Figure 2.8 and
Table 2.7). Thirty ﬁve percent of the 20 primers produced polymorphisms in R4 in
which the primers E, R and T generated unique ampliﬁcation products only in R4, but
not in the controls, and the primers B, D, F, and G generated unique ampliﬁcation
products only in the controls, but not in R4 (Figure 2.9 and Table 2.7). From one to
four unique banding patterns were found in R7 and R4 with each primer (Table 2.7).
These results indicate that in the somaclonal lines, R7 and R4, which demonstrated
higher levels of Fusarium resistance, carried sequence variations unique to the parental
source.

The DNA sequence variations found in R7 and R4 may have been recovered from
either tissue culture because the culturing techniques induced mutations or variant cells
that were already preexisting in somatic tissues. Somaclonal variation could affect
Fusarium resistance in several possible ways. The resistance could be due to mutations
resulting in alternation of gene expression and protein production, for example, changes
in base sequence may result in a more resistant enzyme. Alternatively, increase of the

resistance could be caused by ampliﬁcation of a naturally existing resistant gene in

205:8 05 an “:95.“ c8 8:: E882 05 E “:88: 73:03:28 203 “:5 8an :oueomnaEw
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74

5 80.: 88:: :5 8:: 8228: 05 8 800.08: 388288 0:03 85 0.0an 8:00:88:
053:: 888:0: m3oh: 0::. .98 :09. 88.: :32» 8:2: 3:08: .me 2:803. 88 .8 0:: .8888 0:2 888 05 :5
5: 85:8 0:2 38.: 08 .800. :08 E .8088: 088: a 53 8:82:88: :0: 808 388: 00:2 03: .8 :00. :02: .C. 9 2 80¢
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lawn
v8
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75

Table 2.7. Number of unique ampliﬁcation products that were all present in the resistant
somaclone R7, and present (P) or absent (A) in the resistant somaclone R4 when

compared to 4 control plants.

 

 

 

 

Primer ' No. of unique ampliﬁcation products I No. of RAPD
I Somaclone R7 Somaclone R4 l Proﬁles"
B 1 1 (A) 3
D 1 1 (A) 3
E 0 1 (P) 4
F 1 l (A) 3
G 2 1 (A) 3
H 1 O 3
J 2 O 3
K 2 0 4
L 1 O 4
M 1 0 4
N 1 0 4
O 3 O 4
P 2 O 4
Q 1 0 3
R 1 l (P) 3
T 3 1 (P) 3
Percent of the 75 % 35 %
20 primers
producing
polymorphic bands

 

"RAPD proﬁles were obtained from two different genomic DNA isolations.

20:88 0:: :0 80:: :388: 0:: 8 2:0 880:: 0:03 8:: 82:8:
8:80:38: 088: 88080: 8308 0::. .05: :08 80:: :30:w 2:2: 3:08: .vmm 8:83. :8: .:0 0:0 83:8 0:2 888 0::
:8: S: 83:00 0:2 8:: 0:: .8 :08 8 :08: : .3 88088 88:: 0888 : :23 8:80:38: 20: 80:: 280: 8:2 03: :0 :8
:03 .882: 3:08: .38 8:83. 0: 88:88 :0:3 3: 0:20:88 8:880: 0:: 8 8:2 8888:8820: 058:: dd 0.52,:

 

Imam
:
las—
'88
N W M W ”:23 5.2 ”.2 :

 

77

’Lucullus 234’, although the resistant gene in ’Lucullus 234’ has not been determined.

Resistant somaclonal mutant lines, R7 and R4 described here could provide a source
of increased resistance to F. oxyspomm and F. proliferatwn in an asparagus breeding
program. In further experiments, the resistant somaclonal lines, R7 and R4, could be
crossed with a susceptible asparagus cultivar and their progenies could be checked for
segregation of resistant and susceptible plants to determine if the somaclonal lines have
resistant gene. If a resistant gene exists, the RAPD marker described above may be
useful to isolate the resistance gene by chromosome walking, or as a genetic marker

which can be used in backcross breeding.

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APPENDIX

APPENDIX

The Transformation of Asparagus with A Synthetic Gene Encoding A Novel Lytic

Peptide

ABSTRACT

Cut spear fragments from the monocotyledonous plant Asparagus oﬁ‘icinalis cv.
Lucullus 234 were infected with Agrobacterium tumefaciens strain LBA4404 harboring
an antibacterial and antifungal gene ’Shiva—l’, and two reporter genes GUS and NP’I‘H.
Inoculated tissues selected on kanamycin-containing medium gave rise to antibiotic
resistant calli. Plants were regenerated from the resistant calli and one transgenic plant
(T11) was obtained. This transgenic plant (Tl 1) expressed the GUS marker enzyme and
Southern blot suggested the presence of two copies of Shiva-1 gene integrated into its
genom. Invitro antibacterial assay showed that T11 signiﬁcantly inhibited the growth of
E. coli when compared to a control nontransgenic plant and heat treated T11 indicating
possible production of Shiva-l peptide in T11. T11 was then vegetatively
micropropagated. The micropropagated plants of T11 had signiﬁcantly greater resistance
to Fusarium oxysponan in the greenhouse in comparison with the vegetatively

micropropagated nontransgenic plants.

83

84
INTRODUCTION

The distinctive feature of the Agrobacterium infection process is the natural ability
of this pathogen to export oncogenes and other DNA to a wide range of dicotyledonous
and monocotyledonous plant species (Binns and Thomashow, 1988; Ream, 1989;
Zambryski, 1989; Kado, 1991; Winans, 1992). Agrobacterium is a member of the alpha
subdivision of the class Proteobacteria and gram negative rods which belong to the family
of Rhizobiaceae. It has been divided into ﬁve species: radiobacter, mmefaciens,
rhizogenes, rubi, and viris (Ophel and Kerr, 1990). The Agrobacteriwn which can cause
the crown gall on plants has been classiﬁed as the species A. mmefaciens. When virulent
strains of A. tumefaciens, harboring a large tumor-inducing (Ti) plasmid, infect wounded
plant tissue, a speciﬁc segment of the Ti plasmid, T-DNA, transfers into plant cells and
stably integrates into plant nuclear DNA. Subsequent expression of the T-DNA genes
leads to production of enzymes for biosynthesis of auxin and cytokinin, thereby causing
transformed cells to grow as crown gall tumors and to produce opines which are used as
carbon and nitrogen source by the bacterium for its growth (Petit et al. , 1983). The
infection and T-DN A transmission (transfer and integration) processes involve following
steps: 1) attraction of Agrobacterium cells to the wounded site of plants by chemotactic
agents produced by wounded plants, 2) attachment of Agrobacterium to the plant cells
promoted by polysaccharide controlled by the virulence genes on the chromosome of the
bacterium, 3) induction of expression of speciﬁc vir genes by phenolic compounds
produced by the wounded plants, , 4) generation of a T-DNA copy, 5) Formation of a T-
strand protein complex, 6) movement of the T-complex through the bacterial membranes,

7) targeting of the T—complex into and within the plant cell, 8) targeting of the T-

85

complex to the plant cell nucleus, 9) integration of the T-strand into plant cell DNA
(Zambryski, 1992). T-DN A transmission involves speciﬁc DNA-protein interactions.
The right 23-base-pair direct repeats (Shaw et al. , 1984; Wang et al. , 1984; Peralta and
Ream, 1985; Hepburn and White, 1985) as well as a speciﬁc ﬂanking sequence of T-
DNA (Jen and Chilton, 1986; Peralta et al., 1986; Rubin, 1986; Ji et al., 1988) are
required for efﬁcient transmission of T-DNA. The T-DNA transmission also requires
virulence genes located outside of the T-DNA region on the plasmid (vir) (Garﬁnkel and
Nester, 1980; Klee et al. , 1982; Klee et al. , 1983; Hille et al. , 1984; Stachel and Nester,
1986) and on the Agrobacterium chromosome (chv, psc, exo, art) (Dylan, et al. , 1986;
Cangelosi er al. , 1987; Marks et al. , 1987; Matthysse, 1987; Thomashow et al. , 1987;
Robertson et al. , 1988; Zorreguieta et al. , 1988). The mechanism of the last step of T-
DNA transmission is not yet clear.

Because of this unique feature of Agrobacten’um, the Agrobacterium—plant DNA
transfer system is widely used for plant molecular and genetic engineering experiments.
Numerous genes have been successfully introduced into economically important
agricultural crops. Of these genes, four types have gained worldwide interest as they
improves plant agronomic traits. They are genes conferring: 1). improvement of crop
quality, 2). resistance to insects, 3). resistance to herbicide, 4). resistance to plant
pathogens (disease resistance) including viruses, fungi, and bacteria.

Transgenic tomato, called Flavr Savr, is engineered by antisense gene of ACC
synthase to block ethylene production and delay the onset of fruit ripening. Therefore,
this tomato can ripen on the vine and still be ﬁrm enough to ship so that they take up

more sugar and acids that can improve ﬂavor (Sheeny et al. , 1988; Klee et al. , 1991;

86
Van Brunt, 1992).

A synthetic gene encoding the CryIA(b) protein derived from Bacillus thuringiensis
was introduced into maize. Transgenic maize exhibited excellent resistance to heavy
infestation of European corn borer in the ﬁeld (Koziel er al. , 1993).

Tobacco, potato and tomato were transformed with the bar gene which confers
resistance in Streptomyces hygroscopicus to herbicide of bialaphos. Transgenic plants
showed complete resistance towards high doses of the commercial formulations of
phosphinothricin (PPT) and bialaphos (DeBlock et al. , 1987).

Promising progress have been made with ﬁeld resistance to tobacco mosaic virus in
transgenic tomato (Nelson et al. , 1988) and to cucumber mosaic virus in transgenic
cucumbers (Gonsalves et al. , 1992). Transgenic tobacco, canola, and Brassica napus
with enhanced resistance to fungal pathogen Rhizoctonia solani have been developed
(Broglie et al. , 1991). Transgenic potato expressed an antibacterial gene (Shiva-1) and
increased resistance to bacteria (Dcstefano-Beltran er al. , 1990).

Antibacterial proteins, named cecropins, produced by the giant silk moth (Hyalophra
cecropia) were discovered by a group in Sweden (Boman and Steiner, 1981; Steiner et
al. , 1981; Andreu et al. , 1987). The peptides consist of 35-37 amino acid residues, and
comprise three major forms: A, B, and D. A primary mode of action of the proteins is
membrane disruption and subsequent lysis due to the bacterial’s loss of osmotic integrity
(Christensen et al. , 1988). Additional research has shown that the peptides enhance
overall resistance against fungal diseases and nematodes but normal mammalian and plant
cells are not affected (Jaynes et al. , 1987, 1989a and 1989b; Destefano-Beltran et al. ,

1990). Several analogues of cecropins were synthesized by Dr. Jaynes and associates to

87
test the hypothesis that the biological activity of the peptide is dictated by its physical

structure and not by its primary sequence and to obtain proper expression of Shiva-l in
plants. One analogue in particular, Shiva-1, is derived from cecropin B protein which
has 36 amino acids. Shiva-1 has 47% amino acid homology with Cecropin B, but has
increased biological activity. The size of Shiva-1 gene is 0.125 kb, and it encodes a 38
amino acid long peptide (Destefano-Btran, 1990; Nagpala, 1990).

Cultured stem fragments from A. oﬁ‘icinalis infected by the oncogenic A. tumefaciens
strain C58 harboring the wild-type nopaline Ti plasmid developed tumorous
proliferations. This tissue was propagated in vitro on hormone-free medium. T-DNA-
encoded markers nopaline and agrocinopine were detected in these tissues. However,
transgenic plant was not regenerated (Hernalsteens et al. , 1984). Later, the same group
reported regeneration of transgenic plants of A. oﬁ'icinalis after spear segments were
infected by A. tumefaciens strain C5 8C1 harboring a nononcogenic Ti plasmid-derived
vector containing a chimeric aminoglycoside phosphotransferase [NOS-APH(3’) II] gene
(kanamycin resistant gene) (Bytebier et al. , 1987). Recently, long term embryogenic
callus were infected by nononcogenic A. nunefaciens strain C58 harboring GUS and
NPTII marker genes. One transgenic plant was obtained and conﬁrmed by Southern blot
(Delbreil et al. (1993).

This appendix reports: 1) the development of the transformation of A. oﬁicinalis cv.
Lucullus 234 using the A. twnefaciens strain LBA4404 harboring Shiva-1 gene and
regeneration of a transgenic plant (T11), 2) the veriﬁcation of the transformed plant by
the Fluorometric assay, NPTII Elisa and DNA analysis by Southern blot, 3) test of

possible production of Shiva-1 peptide in T11 by in-vitro antibacterial-assay against E.

88
coli, 4) the evaluation of the vegetatively micropropagated plants of transgenic plant T11

in response to F. oxysporum isolate FOA50 in the greenhouse.

MATERIALS AND METHODS

Plant transformation with Agmbacterium Greenhouse-grown spears of A. oﬁicinalis
L. cv. Lucullus 234 were surface-sterilized for 30 min in a 20% aqueous bleach solution
(5.25 % sodium hypochlorite, Big Chief Bleach, Patterson laboratories, Detroit MI) plus
3mm] per liter of Tween-20 (Sigma Chemical Co., St. Louis MO) and cut into 0.5-cm-
long pieces. The pieces were precultured for 1 day on tobacco feeder plates containing
1 ml of tobacco suspension culture and covered with a piece of sterile Whatman ﬁlter
paper on MSF medium (Elmer et al. , 1989). The spear segments were incubated by
immersing them in an overnight culture at 108 cfu/ml of A. tumefaciens strain LBA4404
for 30 min. This Agrobacteriwn strain contains a pB1121 plasmid that has a Shiva-1
gene and two reporter genes (NPTII gene for kanamycin resistance and GUS gene)
(Figure A. 1) (from Dr. Jaynes, Louisiana State University). The explants were then
blotted dry, replaced on the tobacco feeder plates for a 2 day co-culture period under
dark condition, and transferred to callus-inducing medium (MSF) with 500 mg/l
carbenicillin to kill Agrobacterium and 100 mg/l kanamycin to select transformed tissue
and grown under dark condition for 1 to 2 months. Kanamycin-resistant calli with shoot
primordia were transferred to regeneration medium containing a hormone-free Murashige
& Skoog (MS) medium (1962) supplemented with 500 mg/l carbenicillin, and plants were

regenerated at 27 ° C with 40 nEm'zs'1 light and a 16 h photoperiod. The plants were

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transferred to the greenhouse after acclimazation (Chapter II).

Selection of transformants using Fluorimetric GUS assay and NPTII Elisa
Fluorimetric GUS assay procedure was followed according to Jefferson (1987).
Asparagus plant roots were homogenized in GUS extraction buffer (50 mM NaPO4, pH
7.0, 10 mM deta-Mercaptoethanol, 10 mM NaQEDTA, 0.1% sodium lauryl sarcosine,
0.1% Triton X-100) by grinding in Eppendorf tubes with little disposable pestles. Two
hundred and ﬁfty p1 assay buffer (GUS extraction buffer with 1 mM 4-methyl
umbelliferyl beta-D-glucuronide) was incubated at 37° C to pre-warm and 25 pl of
extract was added to 250 pl GUS assay buffer, 100 p1 mixture were removed, and each
aliquot was added to 0.9 ml stop buffer (0.2 M Na2CO3) at 1, 3, and 5 hour, and
overnight intervals. The methyl umbelliferone (MU) concentration was determined with
Spectro—ﬂuorimeter, excitation at 365 nm, emission at 455 nm.

For the detection and quantitation of neomycin phosphotransferase II (NPT 11) protein
in crude cellular extracts, NPT 11 Elisa kit (5 Prime -> 3 Prime, Inc., Boulder CO) was
used. Asparagus plant shoots were ground in Eppendorf tubes containing 500 p1 of cold
0.25 M Tris-Cl, pH 7.8 and 1 mM phenylmethyl-sulfonyﬂuoride at 4° C. The
homogenates were centrifuged at 7500 x g at 4° C for 30 min, and the protein
concentrations were determined according to Bradford method (Sambrook et al. , 1989).
Two hundred p1 of diluted Coating Antibody were added to each well of microwell
strips, and the microwell strips were covered with paraﬁlm and incubated at 37° C for
2 h. Each well was washed with 1X phosphate buffered saline (PBS) for 3 times, ﬁlled

with 400 pl of IX blocking/dilution buffer, incubated at room temperature for 30 min,

91
and was washed 5 times with 1X washing buffer. Two hundred pl of negative control

and transformed plant extract proteins at concentration of 400 pg/ml were added to the
each well, and the wells were incubated at room temperature for 2 h. Each well was
washed 5 times with 1X washing buffer, ﬁlled with 200 pl of biotinylated antibody to
NPT II, and incubated 1 h at room temperature. The wells were washed 5 times with
1X washing buffer, and 200 p1 of streptavidin conjugated alkaline phosphatase dilution
were added to each well. The wells were incubated 30 min at room temperature. Each
well was washed 5 times with 1X washing buffer, ﬁlled with 200 pl of substrate solution
(5 mg p-nitrophenyl phosphate in 2.5 ml of diethanolamine buffer), incubated at room
temperature for 30 to 40 min in the dark, and read at 415 run against the reagent blank
in a microtiter well reader. All experiments were designed as completely randomized

design and conducted at least two times. Data were subject to ANOVA.

Confirmation of transformed plants by Southern Blot To determine integration of
Shiva-l gene into asparagus plant genome, 5 putative transformed plants (T3, '17, T11,
T13, T16), which showed higher GUS activity or higher NPTII protein when compared
with control plants, were used for Southern blot. Asparagus shoot DNA was isolated
using the procedure of Dellaporta et al. (1985). A. tumefaciens plasmid DNA (pB1121
plasmid) was isolated and puriﬁed from E. coli according to method for large-scale
preparation of plasmid DNA (Sambrook et al. , 1989).

The DNA samples were digested with HindIII at 37°C overnight, and 5 units of
HindIII were used for every 1 pg plant genomic DNA. The digested DNA fragments

were electrophoretically separated on 1.0% agarose gel at 60 voltage for 5 h. The gel

92
was denatured in 1.5 M NaCl and 0.5 M NaOH with agitation for 2 X 15 min, and

neutralized by soaking in 0.5 M Tris, pH 7.4 and 1.5 M NaCl for 2 X 15 min.

The DNA fragments were transferred to Nytran membrane in 10 X SSPE by the
standard capillary blotting. SSPE was prepared according to Sambrook et al. (1989).
After blotting, the membrane was washed in 5 X SSPE for 5 min at room temperature,
dried at room temperature for 30 min and baked under vacuum oven at 80°C for 30 min.

The Shiva-1 probe was made by isolating 1.7 kb Shiva-1 gene fragment from agarose
gels with NA-45 DEAE membrane (Schleicher & Schuell, Lowell MA) according to
Sambrook et al. (1989). The Shiva-1 probe was radioactively labelled with a32P dCTP
using DNA labeling kit (5 Prime -> 3 Prime, Inc., Boulder CO) following Feinberg and
Vogelstein’s method (1983). The labelled probes were then puriﬁed using Sephadex G-
50 column according to Maniatis et al. (1982).

The membrane was prehybridized in a buffer (0.25 ml/cmz) containing 6 X SSPE,
10 X Denhardt’s solution, 0.5% SDS, and 100 pg/ml denatured sonicated salmon sperm
DNA for a minimum of 2 h at 42°C. The prehybridization solution was completely
removed, hybridization solution (0.25 ml/cmz) was added, and the labelled Shiva-l probe
then was added to the solution and mixed thoroughly. The hybridization was carried out
at 65°C for overnight.

The membrane was washed in several hundred milliliters of 2 X SSC and 0.5 % SDS
at room temperature for 5 min, of 2 X SSC and 0.1% SDS at room temperature for 5
min, of 0.1 X SSC and 0.5% SDS for 30 min at 37°C with gentle agitation, and of 0.1
X SSC, 0.5% SDS for 30 to 60 min at 65 °C. The radioactivity in the membrane was

monitored after 15 to 30 min using a-hand—held minimonitor to determine how much

93
longer the membrane need to be washed. The membrane was washed in 0.1 X SSC at

room temperature for 2 min and exposed to X-ray ﬁlm at - 0°C with an intensifying

screen for one to two weeks.

In-vitro anti-bacterial activity assay of transgenic plant T11 To test possible
production of Shiva—l peptide in T11, an anti-bacterial activity assay of T11 was carried
out. The effect of protein extract from T11 and untransformed plants on growth of E.
coli was tested to determine antibacterial activity directly. E. coli strain DHSa was used
in the assay because E. coli is one of the most Shiva-l susceptible bacteria (Nagpala,
1990).

For isolation of the total proteins of T11 and control plant, the roots were
homogenized in 0.01 M phosphate buffer (pH 6.8) and a little sand with a sterile mortar
and pestle on ice. The homogenates were centrifuged for 10 min at 10,000 g, and the
supemants were collected. The protein extracts were sterilized by passing through a 0.22
pm ﬁlter and the protein concentrations were determined at 0.66 mg/ ml according to
Bradford method (Sambrook et al. , 1989).

For anti-bacterial activity assay, the procedure used was a modiﬁcation of the method
of Destefano-Betran, (1991). E. coli bacteria were cultured overnight in 200 ml LB
medium at 37°C and 220 rpm, and the CD. at 600 nm of the culture was determined
using a spectrophotometer to control relative uniform bacterial concentrations from one
assay to another. Twenty pl of the bacterial culture was added to 1 ml of each of the
protein extracts of T11 and control plant, and the heat treated protein extract of T11 by

boiling for 5 min. The treatment of boiling protein extract of T11 was to conﬁrm that

94
the anti-bacterial activity of T1] was possiblely due to the Shiva-l peptide produced in

T11, and not to any possible contaminant. The mixtures were incubated at 37°C and 220
rpm for 1 hour. After the incubation, each mixture was diluted from 101 to 10-5 in 0.01
M phosphate buffer (pH 6.8), and 100 pl of each of the dilutions was cultured on LB
agar medium at 37°C for overnight. The colonies were counted, and percentage of
survival was determined as the numbers of colonies in each treatment / number of
colonies in control. All experiments were conducted as a completely randomized design
with two replicate plates per treatment and were conducted at three different times. All
the data in the table 3.3 were analyzed by ANOVA and Duncan’s Multiple Range Test

was used to separate means.

Evaluation of the vegetatively micropropagated plants of T11 in response to F.
oxysparum in the greenhouse The transgenic plant T11 was vegetatively
micropropagated following the method described in chapter 11 in order to conduct the
screening tests with F. oxysporum. A cultivar of an ornamental asparagus species A.
densiﬂoms, ’Sprengeri’, was used as resistant control and a cultivar of A. oﬁ‘icinalis,
’UC157’, as susceptible control for the screening tests. Twelve of one-month-old
greenhouse-grown transplants of each of the micropropagated transgenic and
nontransgenic plants, and control seedling plants were inoculated with the most virulent
isolate of F. oxysponan (FOA50) using the method of Wacker et al. (1990), and grown
in the greenhouse for 1 month. Plants were fertilized once a week (Peters 20N-20P-20K,
200 ppm). The individual plants were rated for the severity of FOA50 infection

according the visual rating scale of Stephens et al. (1989) and Wacker et al. (1990): 1=

95

healthy plant, no evidence of disease (highly resistant), 2 = few root lesions, < 25 %
diseased (resistant), 3 = moderate number of root lesions, < 50% (moderately
susceptible), 4= many root lesions, <75% (susceptible), and 5 = all roots ﬂaccid or
dead (highly susceptible). The experiments were designed as complete randomized block
design under a natural photoperiod at 25-30°C. Data were subjected to ANOVA and

Duncan’s Multiple Range Test.

RESULTS AND DISCUSSION

Regeneration and selection of transgenic plants After cut spear segments inoculated
with A. tumefaciens LBA4404 were cultured on MSF medium, kanamycin-resistant calli
with shoot primordia developed within 1 to 2 months. Shoots formed on the kanamycin-
resistant calli after 1 to 2 month culture on hormone-free MS medium without
kanamycin. Kanamycin inhibited further shoot development in our experiments. Plants
were further regenerated from the kanamycin-resistant calli on the hormone-free MS
medium, transferred into pots, and grown in the greenhouse.

To screen for transformants, the root tissues of putative transformed and non-
transformed (control) plants grown in greenhouse were tested for GUS activity by
Fluorimetric assay. Signiﬁcantly higher GUS enzymatic activity was obtained in three
putative transformed plants T3, T11 and T13 when compared with control plant (Table
A. 1).

NPTII Elisa was also used to select transformants by detection and quantitation of

NPTII protein level. NPTII protein level was signiﬁcantly higher in three putative

96

Table A.l. Detection and quantitation of GUS activity of putative transgenic plants

(PTP) and non-transformed plants by ﬂuorometric assay.

 

 

 

Plants GUS activity (nM MU/ min/ g fresh root
tissue)l
PTP 11 134.0 A
PTP 13 131.7 A
PTP 3 128.4 A
PTP 7 88.0 AB
PTP 2 87.8 AB
PTP 10 87.6 AB
PTP 12 77.3 B
PTP 9 72.4 B
PTP 4 71.5 B
PTP 6 68.0 B
PTP 1 62.5 B
Non-transgenic plant 50.5 B

 

1Means within a column with different letters are signiﬁcantly different (Duncan’s

multiple range test, P<0.05).

97
transformed plants T1, T7, and T13 than that in non-transformed plants (Table A.2).

One putative transformed plant T13 expressed both GUS and NPTII marker enzymes.
The putative transformed plant T3 and T11 had signiﬁcantly higher GUS activity, but
not NPTII protein. One explanation for this could be that NPTII gene in these two plants
expressed at low level, and NPTII Elisa is not sensitive enough to detect it.
Alternatively, the NPTII gene was not inserted into the genomes of the plants due to
internal deletion of the integrated T—DNA. Internal deletion of the integrated T-DNA in
transgenic petunia has been reported by Deroles and Gardner (1988). Another alternative
explanation could be that NPTII gene was inactivated by methylation. Methylation of
wild type-T-DNA occurred and methylation of regulatory sequences may partially or
fully inactivate gene expression. DNA methylation in plants has been reported and
shown to be inversely correlated to gene expression (Amasino et al. , 1984; Van
Slogteren et al. , 1984). The same explanation could apply to putative transformed plant

T1 and T7 which showed signiﬁcantly higher NPTII protein, but not GUS activity.

Confirmation of transgenic plants To conﬁrm that Shiva-1 gene integrated into
genomes of transgenic plants, ﬁve pre-selected putative transformed plants T3, '17, T11,
T13 and T16 by Fluorimetric assay and NPTII Elisa were analyzed by Southern blot.
Based on the restriction map of pB1121 plasmid (Figure A. 1), it can be predicted that this
plasmid give only one band 1.7 kb in length when digested by HindIII and using the 1.7
kb Shiva-l DNA as a probe. Two bands with the expected 1.7 kb HindIII Shiva-1
containing fragment were obtained in T11 (Figure A.2). No hybridization by 1.7 kb

Shiva-l probe was observed with DNA extracted from either untransformed plant or

98

Table A.2. Detection and quantitation of NPTII protein in crude cellular extracts of

putative transgenic plants (PTP) and non-transgenic plants by NPTII Elisa.

 

Plants NPTII protein(pg)l

PTP 13 18.4 A
PTPl 10.1 B
PTP 7 7.0 BC
PTP 16 5.6 CD
PTP 21 5.3 CDE
PTP 23 3.5 DEF
PTP 18 3.2 DEF
PTP 19 2.9 DEF
PTP 15 DEF
Non-transgenic plant DEF
PTP 9
Non-transgenic plant
PTP l4

PTP 10

PTP 22

PTP 20

PTP 2

PTP l7

PTP 6
Non-transgenic plant
PTP 3

PTP 4

PTP 11
Non-transgenic plant

0 C C O 0 PP
ounmmq:m

mmmmmmmmmmmmmg

OOOOOOOOr-r—r—r—r—H

lMeans within a column with different letters are signiﬁcantly different (Duncan’s

multiple range test, P<0.05).

 

Figure A.2. Autoradiograrn of Southern blot of DNA extracted from transformed and
untransformed plants, digested with restriction enzyme HindIII and membrane hybridized
with 1.7 kb Shiva-l probe. Lane 1 to 4: transformed plants T11, T7, T13 and T16.
Lane 6: untransformed plant (control).

100

other putative transformed plants (Figure A.2). The occurrence of two bands in T11
may be caused by two integrations of Shiva-1 gene in two loci in one chromosome or in
different chromosomes with rearrangement of the T-DNA and/ or methylation of the T-
DNA sequences especially those in HindIII sites. Truncation, rearrangement, repetition
or methylation of the introduced T-DNA have been reported in transgenic petunias (Jones
et al., 1987; Deroles and Gardner, 1988) and tobacco (Hobbs et al., 1990). One
putative transformed plant T13 had signiﬁcantly higher GUS activity and NPTII protein
level with comparison to untransformed plants, but no hybridization with Shiva-1 probe.
No presence of Shiva-l gene in T3, '17, T13 and T16 may be due to internal deletion of
the integrated T-DNA. Deroles and Gardner (1988) reported internal deletion of the
integrated T-DNA in transgenic petunia.

Among 16 southern blots of transgenic plants, 2 blots showed expected 1.7 kb
HindIII Shiva-1 DNA fragment band in transgenic plant T11. No hybridization by 1.7
kb Shiva-a probe was observed with pB1121 plasmid DNA (positive control) which
contains Shiva-l gene in one blot (data not shown in Figure A.2). This may be
explained that the concentration of the plasmid DNA was too low to be detected by this

method.

Antibacterial activity of transgenic plant T11 The protein extract from T11
signiﬁcantly prohibited bacterial growth when compared with untransformed plant extract
(Table A.3). Sixty ﬁve percent of bacteria survived after they were treated with the
protein extract from T11 (Table A.3). The protein extract at concentration of 0.66

mg/ ml from T11 had the antibacterial activity equivalent to 0.6 pM pure Shiva-1 peptide

101

Table A.3. Inhibition of E. coli growth by crude protein extracts from transgenic plant

 

 

 

 

T11.

Treatment Average number Percent of
of surviving surviving
bacterial bacteria

Protein extract 29 A 65.9 A

of transgenic plant 11 (I‘ll)

(0.66 mg/ ml)

Protein extract 44 B 100 B

of non-transgenic plant (CK)

(0.66 mg/ ml)

Boiling protein extract of 41 B 93.2 B

transgenic plant T11
(0.66 mg/ml)

 

1Means of 2 replicate plates from three replicate experiments, means within a column

with different letters are signiﬁcantly different (Duncan’s multiple range test, P< 0.05).

102
(Nagpala, 1990). The inhibitory effect of the protein extract from T11 on the bacterial

growth was almost completely overcome by boiling the protein extract of T11 (Table

A.3). These data indicate that Shiva-l peptide may be produced in T11.

Evaluation of the vegetatively micropropagated plants of T11 in response to F.
oxysporum in the greenhouse After vegetatively micropropagated plants of each of T11
and nontransgenic plant were screened with FOA50, the transgenic plants were found to
be signiﬁcantly more resistant to FOA50 than the nontransgenic plants (Table A.4). Of
12 transgenic plants, 58.3% of them were rated at a 1 or 2, whereas 83.3% of
nontransgenic plants were rated at a 3 or 4 (Table A.4).

Southern blot suggested that the transgenic plant T11 had two copies of Shiva-l gene
integrated into its genome. In vitro antibacterial activity assay indicated possible
production of Shiva—1 peptide in T11. The transgenic plants from vegetative
micropropagation showed a signiﬁcant increase in resistance to FOA50 in the
greenhouse. The Shiva-1 gene is under the control of the wound-inducible potato
proteinase inhibitor H promoter. The proteinase inhibitor 11 promoter is usually turned
on by severe wounding through mechanical means or insect injury (Thornburg et al. ,
1987). F. oxysporum enters plants by directly penetrating meristimatic zones of roots
using appressoria, by partially digesting root tissue using its enzyme, or through crushed
cortical cells by emerging lateral roots (Nelson et al., 1981). It is possible that this
promoter was activated by fungal invasion through fungus induced wounding.
Transgenic potato plants with Shiva-1 gene under the control of the same promoter

showed that the expression of Shiva-1 gene is triggered by mechanical and pathogen

103

Table A.4. Disease rating of vegetatively micropropagated plants of transgenic plant
T11 and nontransgenic plants, and control plants screened with Fusarium oxyspor um

(isolate FOA50) in the greenhouse.

 

 

 

 

 

 

 

Plant source No. of plants in disease rating Mean

scales" disease
' y

l 2 I 3 4 I 5 mung

’UC157’ 0 2 8 2 0 3.0 A

(Susceptible control)

Micropropagated seed plants 1 1 7 3 0 3 .0 A

of ’Lucullus 234’

Micropropagated plants 4 3 4 1 0 2 .2 B

of transgenic plant T11

’Sprengeri’ 11 1 0 0 0 1.1 C

(Resistant control)

 

"Twelve plants of each of plant sources were screened with F OA50 using complete
randomized block design. Disease was assessed using a visual rating scale: 1= no
disease, 2= < 25% diseased, 3= < 50% diseased, 4= <75% diseased, 5= >75%
diseased.

Means of disease rating within a column with different letters are signiﬁcantly different

(Duncan’s multiple range test, P < 0.05).

104
induced wounding (Destefano—Beltran et al. , 1990).

Asparagus transgenic plants from vegetative micropropagation with enhanced
resistance to F. oxysponan may be useful for asparagus breeding. Clearly, futher
analysis such as Nothern blot and Western blot are necessary to study expression of

Shiva-1 gene in T11 and to determine mechanism of the resistance.

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