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This is to certify that the
thesis entitled
EVALUATION OF NATURAL AND ENGINEERED RESISTANCE

MECHANISMS IN SOLANUM TUBEROSUM L. FOR RESISTANCE
TO PHTHORIMAEA OPERCULELLA ZELLER

 

 

presented by

Andrea Lynn Westedt

has been accepted towards fulfillment
of the requirements for

Masters degree in Crop & Soil Sciences/
Plant Breeding & Genetics

Major professor

Date EN 2717/???

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

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MSU Is An Affirmative Action/Emu Opportunity Intuition
Walnut

 

EVALUATION OF NATURAL AND ENGINEERED RESISTANCE MECHANISMS
IN SOLANUM TUBEROSUM L. FOR RESITANCE TO PHTHORIMAEA
OPERCULELLA ZELLER
BY

Andrea Lynn Westedt

A DISSERTATION

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

MASTER OF SCIENCE
Department of Crop and Soil Science

1997

ABSTRACT

EVALUATION OF NATURAL AND ENGINEERED RESISTANCE MECHANISMS
IN SOLANUM TUBEROSUM L. FOR RESITANCE TO PHTHORIMAEA
OPERCULELLA ZELLER

BY
Andrea Lynn Westedt

Potato tuber moth (Phtborimaea operculella Zeller) is a
highly destructive pest of cultivated potato (Solanum
tuberosum L.) that is responsible for damage to both leaf and
tuber tissues. Bacillus thuringiensis (Bt) is an effective
control measure and has only minimal persistence when
topically applied. Host plant resistance (HPR) is a central
component to developing an IPM program to control potato tuber
moth. This research 1) tested the efficacy of a codon-
modified CryV-Bt gene constitutively expressed in potato, and
2) combined the CryV-Bt expression with natural HPR. Lemhi
Russet, and two lines with host plant resistance mechanisms,
USDA8380-1 and L235-4, were transformed with the CryV-Bt.gene.
Transformation was confirmed by polymerase chain reaction and
Southern analysis. Potato tuber moth bioassays were conducted
to test the efficacy of the introduced gene. These assays
showed that high levels of expression occurred in the CryV-Bt
transgenic lines, with up to 96% potato tuber moth control.
These transgenic lines provide a germplasm base to examine

combined insect-resistance mechanisms to achieve durable HPR.

ACKNOWLEDGMENTS

I would like to express my appreciation to Dr. Dave Douches
for his guidance and assistance throughout my thesis work. I
would also like to thank.guidance committee members Dr. Edward
Grafius, and Dr. Kenneth Sink for their assistance with this
research project.

I am also grateful to my roommate and fellow graduate
student Dave Maas for all his support and help. Thanks also
go to Peter Hudy, Chris Long, Raphael Mendez, Kim‘Walters, and
Kelly Zarka, and all the undergraduates that have assisted me
in the laboratory studies.

I would especially like to thank.my parents without whose

continued support I wouldn't have achieved all that I have.

iii

TABLE OF CONTENTS

INTRODUCTION .............................................. 1
Potato Tuber Moth ............................ ........1
Host Plant Resistance ........................ . ....... 3
Bacillus Thuringiensis.... ................ .... ....... 7
Genetic Engineering...... ............................ 9
Objective.. ........................ .... .............. 10
MATERIALS AND METHODS ............. ........ ............ . ..12
Plant Materials ...................................... 12
Transformation protocol..............................12
DNA Quickprep Isolation procedure .................... 16
Polymerase Chain Reaction.. .......................... 18
Potato Tubermoth Rearing ............................. 19
Potato tuber moth bioassay ......... . ..... ............19
Southern Blot ........................................ 23
Northern Blot ............................... .........24
RESULTS ................................................... 25
Plant transformation ......................... ........25
Potato tuber moth bioassays .......................... 26
Southern and northern analysis ....... . ............... 30
DISCUSSION..... ........................................... 39
LITERATURE CITED .................................. . ...... .47
APPENDIX A ................................................ 52

iv

LIST OF TABLES

Table 1. Components of potato nodal culture propagation
medium ...................... ... ..... . ................ 13
Table 2. Yadav and Sticklen potato shoot regeneration medium
for explants...... ........... .......... ..... .........14
Table 3. Ty-broth... ...... .... ................. . .......... 17
Table 4. Primers used in PCR analysis of putative
transgenics.... .......... . ................. . ......... 21
Table 5. Polymerase Chain Reaction mix .................... 22
Table 6. Means of potato tuber moth preliminary tests of
Lemhi Russet transgenic lines in detached leaf
bioassays .................... . ....................... 28
Table 7. Means and standard deviations of Lemhi Russet for
potato tuber moth mortality of potato tuber
moth ................................................. 29
Table 8. Means for potato tuber moth mortality preliminary
trials of 8380-1 Cry-Bt lines and untransformed 8380-1
in detached leaf bioassays ........................... 32
Table 9. Means for potato tuber moth mortality second
preliminary trials of 8380-1 Cry-Bt lines and
untransformed 8380-1 in detached leaf bioassays ...... 33
Table 10. Means and Standard deviations of 8380-1 for insect
mortality ............... ..... ...... . ................. 34

Table 11. Means and standard deviations of L235—4 for insect

mortality.................................... ........ 35
Table 12. Lines used for Southern and northern analysis...38
Table 13. Summary of morbidity scores for Lemhi Russet,

L235-4, and USDA 80-1 ........................... . . . . . 52

vi

LIST OF FIGURES

Figure 1. PCR Results of Lemhi Russet.....................27

Figure 2. Southern analysis of transgenic line probed with
the CryV-Bt gene................................36

Figure 3 Northern analysis of CryV-Bt transgenic lines

probed with CryV—Bt/Gus gene .................... 37

vii

INTRODUCTION

First introduced into the United States in 1719, the
cultivated potato (Solanum tuberosum subsp. tuberosum L.)is
now the 4th most important crop worldwide. The potato is
widely grown over many latitudes and elevations in 130 of the
world's 167 independent countries. It is a nutritionally
dense crop used for human consumption, livestock feed,
distillation products, processed into starch, paste dye, or
converted to ethanol (Rhoades 1982).

There are many constraints to potato production
including a wide array of insects, viruses, diseases, and
abiotic stresses. One of the most important insect pests in
potatoes worldwide is the potato tuber moth (Phthorimaea
operculella Zeller). Warm geographic regions with average
daily temperatures reaching 16%:or greater incur the greatest

damages by potato tuber moth (Raman & Palacios, 1982).

Potato tuber moth

Potato tuber moth, Phthorimaea operculella Zeller is
wide-spread but, is generally recognized as originating in
South America where the potato alsoloriginated (Goldson et al.
1985). This pest is of greatest importance in warm climates
with average daily temperatures of 16%L

The tuber moth causes damage in both field and storage

(Raman et al. 1982; Ebora et a1. 1994). All developmental

2
stages except the eggs are killed by exposure to -6.7W: and
eggs are also killed by exposure to temperatures below 1.7-
4wwmrfor four months. In addition, all other life stages are
destroyed by temperatures in excess of 36°C for a 15 day
period. (Trivedi et a1. 1992).

Potato tuber moth attacks the potato by mining in the
leaves and/or tubers. This damage is caused only by the
larval stages (Raman 1980; Trivedi et al. 1992; Goldson &
Emberson 1985). Larvae penetrate the leaves and feed within
them as well as tunneling within leaf veins and stems of the
plant. This damage causes loss of leaf tissue, death of
growing points and.weakening or breakage of stems (Raman 1980,
Bald & Helson 1944). The tubers are infested by eggs
deposited on the surface of the soil near the stem and those
laid near the tuber eyes when in storage. The larvae mine
into the tuber causing irregular tunnels both near the surface
and deep inside the tuber rendering them unfit for human
consumption. Potato tuber moth is not strictly confined to
potatoes. When potatos are not avalible, its feeding habits
may extend to numerous Solanaceae species including tobacco
(Nicotiana tabacum L.), tomato (Lycospersicon esculentum
Mill.) and eggplant (Solanum .melongena var.) (Goldson &
Emberson 1985). The potato tuber moth is unable to rapidly
multiply if confined to leaf mining alone (Trivedi et al.
1992).

The life cycle of potato tuber moth includes egg, larvae,

3
pupae and adult stages. The cycle may' be completed within 20-
30 days at 28°C. There may be as few as two generations and as
many as twelve generations in a single year (Raman 1980). The
0.5mm eggs, when laid, are initially pearly-white and
gradually darken to black just before hatching. They tend to
be laid in groups on rough surfaces (Goldson et al. 1985).
Eggs may be deposited in several places, such as the underside
of leaves or stems, proximal to tuber eyes, on sacks and
containers used for storage, and dirt or debris on or between
tubers (Raman 1980). Duration of incubation varies with
temperature, but generally takes 5 days (Goldson et al. 1985)
The larvae are about 1mm at emergence, 10mm long when fully
grown, and white to yellow in color with a dark brown head.
This stage takes about eight days depending on *weather
conditions. Pupae are brownish, 6mm long and enclosed in a
white silk cocoon. They may be in various places such as on
old dry leaves on the plant, storage walls, tubers, sacks,
containers, rubbish on the storage floor, old damaged tubers,
in soil litter, and eyes of tubers (Raman 1980). Adults have
a wing span of about 15mm and have a silvery body with gray-
brown fore wings and dirty white hind wings. The adults live

for 10-15 days (Goldson et. a1 1985; Raman 1980).

Host plant resistance
Host plant resistance is a desired central component of

an integrated pest management program (IPM)to control the

4

tuber moth. Two major forms of host plant resistance,
antixenosis and antibiosis have been described for potato.
Antixenosis acts to deter insects from colonizing a given
plant by the presence of physical impediments (Kogan 1982).
Antibiosis, encompasses all adverse effects of a temporary or
permanent nature resulting from the ingestion of plant
material by an insect (Kogan 1982).

A form of morphological antixenosis is a structural
feature which impairs the normal processes of insects (Kogan
1982). Three wild potato species have been found to have high
densities of glandular trichomes. They are S.berthaultii, S.
polyadenium, and S. tarijense (Tingey 1984). The defensive
system of S. berthaultii results in small-bodied insects
exhibiting modified behavior, including host avoidance and
restlessness, reduced feeding, delayed development, and
diminished longevity (Tingey 1991).

There are two types of glandular trichomes: Type A
glandular trichome is 120 to 210 mu in length bearing a 50 to
70 mu diameter, four-lobed, membrane-bound head. Type B
glandular trichomes are 600 to 950 mu and bear a 20 to 60 mu
ovoid droplet at the tip (Gibson 1971, Tingey 1991). When the
head of a type A trichome is manually ruptured by an insect,
a clear exudate is released. Once this clear, water-soluble
material comes in contact with the atmosphere it reacts with
oxygen and oxidizes to a black, insoluble material that

collects on the underside of insect pests eventually causing

5

immobility and death (Gibson 1971). Type A trichomes operate
via the enzymatic activities. of an (L-requiring oxidase
(Steffens et al. 1991). Once the membrane of type A trichomes
are ruptured and once the oxidase is released it no longer
produces secretions. This is in contrast to the type B
trichome which continually produces a small amount of sugar
esters (Steffens 1991).

Incorporating this resistance has been difficult because
of tight linkage groups for trichome traits and poor agronomic
traits in S. berthaultii (Kalazich et al. 1991). This is
demonstrated by the strong association of the presence of type
B trichomes and undesirable agronomic traits in backcross
generations with S. tuberosum, but no association was found
among intercrosses. Transmission of type B trichome exudate
is more complex than type A trichome traits (Kalazich et al.
1991). There is indication that a short cycle through callus
culture may help to introgress trichome resistance (Lentini et
al. 1990). At the diploid level the presence of the B
trichome is controlled by a single dominant gene, but in
crosses with S.tuberosum, at least one set of recessive genes
is also necessary for optimum secretory capacity (Tingey et
al. 1984; Kalazich et al. 1991).

Reduced pupation has been attributed to antibiosis in
potato tubers (Raman & Palacios 1982). Chavez, et al. (1988)
reported that this type of resistance to potato tuber moth was

transferred to all progenies except those hybrids in which S.

6

tarijense was the resistant parent. Glycoalkaloids are the
most common form of antibiosis in potato (Sinden et al. 1986).
Steroid.glycoalkaloids (solanine:and.chaconine) are present in
all potato tubers and_ processed. products (Sinden 1987).
Glycoalkaloids below 20 mg/100g (mg %) fresh weight are
considered safe for human consumption. S. tuberosum generally
contains only 2-10 mg% glycoalkaloid, however, these levels
are greatly influenced by genetic and environmental
conditions. Factors such as soil type, soil moisture,
fertilizer level and pesticides, light quality and quantity,
and mechanical damage all may contribute to increased levels
of glycoalkaloids in the tuber. Wild potato species such as
S. chacoense and.S. commersonii can have concentrations of 230
mg % and 500 mg % of glycoalkaloids, respectively (Sinden
1987).

Glycoalkaloid synthesis is highly heritable and appears
to be a quantitative rather than qualitative trait (Sinden
1987). The high glycoalkaloid content of wild species can be
transmitted to hybrids with S. tuberosum causing the progeny
to be unfit for human consumption (Sinden 1987). Acetylated
glycoalkaloids are the most potent form of glycoalkaloids
present in potato. Leptines are acetylated analogs of the
common potato glycoalkaloids, solanine and chaconine.
Leptines and.other acetylated glycoalkaloids are only'reported
to be synthesized by some accessions S. chacoense and

synthesis only takes place in the leaves (Sanford et al.

7
1996). It was found in clone USDA 8380-1 that leptine and
total glycoalkaloid (TGA) content differed little between
diploid and tetraploid forms.

Glycoalkaloid synthesis has been demonstrated to have a
high heritability which can be exploited by breeders to
develop host plant resistance in commercially acceptable
germplasm (Sinden 1987). Crosses have been made between S.
tuberosum and S. chacoense producing TGA levels as high as
1698 mg % with 89-91 % being leptines. Only small amounts of
solanine and chaconine were produced- Leptines were not found
in the tubers of the 136 tested hybrids. The levels of
solanine and chaconine at rates of 30 to 100 mg % in the
tubers were considerably higher than normal rates for S.

tuberosum (Sanford et al. 1996).

Bacillus thuringiensis

Bacillus thuringiensis (Bt) is an aerobic, gram-
positive, soil bacterium that accumulates high levels of
insecticidal crystal proteins during sporulation (McGaughey et
al. 1992; Barton et al. 1993). These crystalline protein
inclusions, or 6-endotoxins, are the principle active
ingredients in Bt formulations currently in use (McGaughey et
al. 1992). These crystal proteins are processed in the gut
juice of susceptible insects yielding active 6-endotoxins.
The molecular mechanism of the toxicity of the 6-endotoxins is

composed of a binding phase which seems to determine the host

8

specificity, and is ultimately responsible for membrane
disruption (Parenti 1995). The advantage of the Bt toxin over
conventional chemical insecticides is order specificity for
insect pests (MCGaughey 1992). The original insecticidal
protein has a specificity for lepidopteran insects. This
specificity makes it generally recognized as ecologically
sound. These toxins have no known detrimental effects on
mammals or birds (McGaughey et al. 1992). Topically applied
Bt (i.e.Dipel), has been used with some success for 20 years.
However, its poor persistence results in a need for multiple
applications (Barton et al. 1987).

The Bt toxin gene has been cloned, codon-modified and
inserted in various crop species (Barton & Miller 1993).
Since the initial cloning work, other Bt genes also have been
isolated and cloned, that have other insect specificities for
coelopteran and dipteran orders. Transformations of potato
with.a wild type Cry IA Bt toxin gene have produced low levels
of Bt expression with 20-60% insect mortality at the
laboratory level (Peter Hudy, personal communication). These
levels of expression for wild type genes are consistent with
previous results (Barton et al. 1987; Ebora et al. 1994; and
Wfinn et al. 1996). The Cry V Bt gene, effective against both
lepidOpteran and coleopteran insects has been codon-modified
to increase expression in plants (M. Wilson, ICI Seeds
personal communication) . The efficacy of other codon-modified

Bt genes such as Cry I and Cry III Bt have been found to be

9
greater than the wild type Bt in crop plants (Perlak et al.

1990; Wfinn et al. 1996).

Genetic engineering

Transgenic plants are plants into which foreign genes
have been introduced (Martin 1994). First generation
applications of genetic engineering to crop plants are
targeting the same issues currently being addressed by
traditional breeding: 1) improved production efficiency, 2)
increased market focus, 3) enhanced environmental
conservation, and 4) pest resistance (Gasser & Fraley 1989).
This new technology is not viewed as replacing but rather
complementing conventional plant breeding (Martin 1994).
Transgenic plants have to be evaluated in much.the same manner
as new selections. At present, genetic engineering is limited
to traits controlled by one or very few genes (Martin 1994).

The primary vector for plant transformation involves the
use of the Agrobacterium tumefaciens (White 1993).
Alternative methods can be divided into two types of
approaches, direct physical and transmission of genetic
material by modified plant viruses (White 1993). These
alternative methods include: high-velocity microprojection,
gene transfer into pollen, direct injection into reproductive
organs, microinjection into cells of immature embryos,
rehydration of desiccated embryos (Gasser & Fraley 1989),

protoplast transformation, and electroporation (White 1993).

10

Due to the relative ease of tissue culture regeneration
of potato many methods of transformation have been used on
them. Most genetically engineered potatoes have been produced
using disarmed A. tumefaciens from which the gall forming
genes have been removed (Martin 1994). The tumor inducing
genes are replaced with the genes of interest and the bacteria
then transfer this gene to the plant. The transfer section of
DNA, or T-DNA is delineated by almost perfect 25 bp direct
repeats. The gene required for the transfer of the DNA from
the bacteria to the plant is known as Ti (tumor inducing)
region on an separate plasmid than the T-DNA. Transformation
utilizing A.tumefaciens makes use of cointegrated or binary
vectors. Cointegrated vectors have the gene of interest
integrated on the same plasmid of the Ti region. In the
binary system the T-DNA is on a small separate plasmid from
the Ti plasmid. The advantage to the binary system is that it
provides a small plasmid that is relatively easy to
manipulate. Once the plant tissue has been exposed to the
bacteria it is placed in an aseptic tissue culture environment
to:regenerate plantlets from single transformed cells. (Martin

1994, Gasser & Fraley 1989, White 1993).

Objective
The overall objective of this research was to combine
natural resistance mechanisms with the CryV-Bt gene, via

transformation, to confer host plant resistance to the potato

ll
tuber moth. These plants provide an opportunity to test if
gene pyramiding leads to a more durable host plant resistance.
The specific research objectives were: 1) obtain transgenic
plants which express the CryV-Bt toxin gene, 2) determine the
strength of the natural resistance mechanisms in the
untransformed plants against potato tuber moth, and 3)
evaluate the pyramiding of the CryV-Bt gene together with

glandular trichomes or foliar leptines.

MATERIALS AND METHODS

Plant materials:

The potato plants utilized for transformation with the CryV-Bt
gene were USDA 8380-1, a S. chacoense selection obtained from
USDA/ARS Beltsville; L235-4, from Cornell University (Plaisted
et al. 1992), and the cultivar Lemhi Russet. Two of these
genotypes were chosen because of their natural host plant
resistance factors: 8380-1 (leptines) and L235-4 (glandular
trichomes). Lemhi Russet is a potato tuber moth susceptible
commercial variety and has demonstrated high regeneration

ability in tissue culture.

Transformation protocol:

Stock plants of three genotypes were micropropagated via
shoot-tip, four plants per GA-7 Magenta vessel using Murashige
and Skoog (1962) modified medium listed in Table 1.
Environmental conditions were a 16-h photoperiod under
florescent lights (30 MB m'2 s") at 23° C for all stages.

The Yadav and Sticklen (1995) plant regeneration protocol
was utilized for the Agrobacterium-mediated transformation
scheme. Leaves from the stock plants were removed and tips
and petiole ends cut to produce 5-10 mm wide strips. The

leaves were placed abaxial-side down onto step-I medium (Table

12

13

Table 1. Components of the potato nodal culture propagation
medium.

 

INGREDIENT Amount per liter
MS Salts (Sigma M-5524) 4.3 g/l
MS Vitamins (Sigma M-6896) 1.0 ml/l
GA3(.25mg/ml stock) 1.0 ml/l
Ca Pantothenic Acid (2.0 mg/l) 1.0 ml/l
Sucrose 30.0 g/l

pH to 5.7 with NaOH (1N)
Bacto Agar 7.0 g/l

 

14

Table 2. Yadav and Sticklen (1995) potato shoot regeneration
medium for leaf explants (per liter).

 

 

INGREDIENTS STEP I STEP II
MS-M5519 (Sigma number) 4.4 g 4.4g
Thiamine-HCl (1mg/ml stock) 0.9ml 0.9ml
Sucrose 30.0g 30.09
Trans-Zeatin (0.5 mg/ml stock) 1.0ml 1.0ml
2,4 D (2mg/ml stock) 1.0ml None
GA3 (2mg/ml stock) None 1.0ml
pH to 5.7-5.8
Bacto Agar 7.0g 7.0g
Autoclave
Timentin (200mg/ml stock) None 1.0 ml
Kanamycin-SQ,(50 mg/ml stock) None 1.0 ml

 

15

2) and precultured for four days.

A. tumefaciens was grown for two days in 10 ml Ty
broth (Beringer 1974) with 50 mg/ml kanamycin (Table 3) in
each 50 ml Erlenmeyer flask at 1800 rpm and 28°C. One
milliter of this mixture was removed and placed in 50 ml of
Ty-broth in a 250 ml Erlenmeyer flask and grown for 6 h at
28°C. The 50 ml Ty-broth solution was used directly for
inoculation of the leaves“ Ten leaves were inoculated with A.
tumefaciens for 3-10 minutes in 15ml of Ty-broth solution.
The leaves were then blotted on sterile singlefold paper (Fort
Howard Co., Green Bay, WI) and placed on fresh step I-medium
for two days. Environmental conditions were a 16-h
photoperiod under florescent lights (30 uE m’2 s") at 23°C for
all stages. After the two days co-cultivation, the leaves
then were washed in liquid step II-medium of Yadav and
Sticklen (1995) (Table 2). The leaves were then blotted on
sterile singlefold paper and.placed.abaxial-side.down on solid
step II-medium. The leaf disks were transferred to fresh step
II-medium every two weeks. One transformation experiment of
eight plates with five leaf pieces per plate were run for
Lemhi Russet and 8380-1, while two transformation experements
were completed on L235-4.

Healthy shoots were excised when 2-3 mm in length and
transferred to selection medium (nodal propagation medium
containing 50mg/1 kanamycin) in 25X100mm culture tubes.

Environmental conditions were a 16-h photoperiod under

16
fluorescent lights (30 uE-m‘z-s") at 23°C for all stages.
Individual shoots that.readily rooted.in selection mediumwwere
given a line code and propagated for studies confirming gene

integration.

DNA Quickprep Isolation Procedure:

Two 2.5 mm leaf disks from. rooted lines, putative
transgenics, were collected for DNA isolation. The DNA
extraction protocol used was a modified version of Edwards et
al. (1991). The tissue was collected by punching out two leaf
disks with the lid of a sterile, 1.5 m1 Eppendorf tube, or two
leaves of approximately 2.5mm size, were removed from the
tissue culture plants. 400 ul of CTAB extraction buffer (2%
CTAB, 1.4 M NaCl, 0.2 M EDTA, 0.1 M Tris-Hcl) with 1% B-
mercaptoethanol added to the leaf tissue. The tissue was
completely macerated. with a sterilized. pestle which. was
attached to a mechanical drill in a 1.5 ml Eppendorf tube.
The pestles were washed. with 70% EtOH between samples.
Samples were then incubated at 65%: for 30 minutes. After
incubation, 400ul of chloroform:isoamyl alcohol (24:1) was
added to each sample tube, mixed by inversion for one minute,
and then centrifuged for 5 min. at 15,000 rpm. The top
aqueous layer was transferred to a new 1.5 ml Eppendorf tube
and 400111 of ice cold isopropanol was added solution to
precipitate the DNA. The tubes were then centrifuged for 5

min. at 15,000 rpm. Supernatant was discarded and the tubes

17

Table 3. Ty-broth (Beringer 1974).

 

 

INGREDIENT (for one liter) LIQUID SOLID
Tryptone 5.0g 5.09
Yeast Extract 3.0g 3.09
CaCl-ZHR) 0.59 0.59

Bacto Agar

15.09

18
were dried under vacuum for 10 minutes at 25%:. The DNA was
resuspended in 100 pl of sterile millipore water with 4pl of
Rnase A (10 mg/ml stock solution) and were left standing for
15 min. at 25%:. Following the RNase treatment, the DNA was
precipitated with 300pl of ice cold 100% EtOI-I. Tubes were
centrifuged at 15,000 rpm for 10 min., the supernatant was
poured off and the DNA was dried under vacuum. Tubes were
dried in a speed-vac for 10 min. then resuspended in 50 pl of
sterile millipore filtered (0.22 microns) water. The DNA was
quantified utilizing fluorometer model TK0100 (Hoeffer

Scientific, San Fernando, California).

Polymerase Chain Reaction (PCR):

DNA was amplified according to Gibco BRL (Bethesda, MD)
with the primers described in Table 4. Gibco Taq DNA,
buffers, and dNTP mixtures were used for the PCR reaction
according to Gibco recommendations. The reaction components
were added to a sterile 0.5-ml microfuge tube (see Table 5).

PCR components were briefly centrifuged and then overlaid
with 50pl of sterile mineral oil. The amplification
conditions were as follows: 1 cycle = (4 min. @ 94%: ); 40
cycles = ( 1 min. @ 94°C, 1 min @58°C, 1.5 min @ 72°C ). Once
completed, a 30 pl aliquot of the PCR reactin mix was run on
a 1% agarose gel stained with 8.5 p1 ethidium bromide

(10mg/ml) and run at 25 volts for 18 h.

19
Potato Tuber moth Rearing:
Egg Production:

Tuber moth pupae were placed into a 1 gallon glass jar
containing a 50% honey/water solution in a 36 ml souffel cup
as a food source, which was present throughout the egg laying
period. The glass jar was covered with nylon window screen
and sealed with a rubber band to provide a landing surface for
oviposition. A No.1 150 mm Whatman filter paper was placed
over the screen to serve as a surface for egg laying. The
cultures were maintained at zgrtin complete darkness.

Larva Through Pupa Development:

The filter papers containing eggs were collected every
two days and placed on disease-free tubers or utilized for
feeding studies. The tubers were kept in a plastic tub (45 x
30 x 20 cm3) at 26°C in complete darkness. Corrugated
cardboard, cut into 5 cm squares and stacked five high to
provide six suitable pupal chambers. 'The tub was covered.with
a double layer of cheesecloth.to prevent escape of larvae, but
still allow air exchange. The newly hatched larvae were
allowed to mine the tubers for a food source. Fully developed
larvae would enter the pupal chambers. Pupae were collected

and moved to one gallon glass jars to continue the process.

Potato tuber moth Bioassay:
All putative transgenic lines, those that passed the

rooting assay and were PCR-positive, were maintained in tissue

20
culture. Two set of such plants were kept in tissue culture,
and a third plant was transferred to the greenhouse and grown
for DNA and RNA extractions as well as for use in insect
feeding studies.

A detached leaf bioassay was used to test for feeding
efficacy of the Cry V-Bt gene. Leaf tissue was collected in
the morning by cutting off a young fully expanded leaf. The
petiole of the detached fully expanded leaf was cut off under
water using a new single edged industrial razor blade. The
petiole was inserted in a pre-moistened half sponge 1 cm in
diameter by 1 cm long, and then placed in a glass vial full of
water. The unit was then placed on a pre-moistened 150 mm
diameter filter paper disk labeled with the genotype.

All lines including a negative control were tested in
preliminary bioassays using two trialS‘with five first instars
per leaf. The larvae were placed on the leaves near the mid-
rdjn The 150 X 20 mm disposable petri dishes were covered and
set on a 25 pE m'2 s‘1 lighted lab bench kept at 25°C:|:2, for 48
h when health and mortality records of the potato tuber moth
larvae was recorded. Percent mortality was calculated as:
(number alive after 48h/1arvae)*100. Missing larvae were
considered dead. An index of health was assessed on a visual
scale of "1-5", with "0" being dead and "5" normal. Scores of
"1-4" were based on relative health of those left alive on the
leaves. A score of "1" represented all near death, "2" one

larvae may have been growing, "3" all growing, but not at

21

Table 4. Primers used in PCR analysis of putative transgenic
potato lines.

 

 

Primers Sequence (5’-3') Total PCR- Molecular
Bases Amplified. Weight
Fragment
Size
Cry V AACTGGAGGTCGGTGGTGC 25 684 bp 7793
Forward. TGGCGT
Cry V GGACCATCGGCGGCACCCT 25 7576
Reverse CAACAT
NPTII CGCAGGTTCTCCGGCCCGC 26 255 hp 8001
Forward TTGGGTG
NPTII AGCAGCCAGTCCCTTCCCG 25 7535
Reverse CTTCAG

 

22

Table 5. Polymerase Chain Reaction mix. Buffers, Taq DNA
Poloymerace, MgCl, and DNTP mixture from Gibco BRL (Bethesda,

 

 

MD).
COMPONENTS VOLUME FINAL CONCENTRATION
10X PCR buffer 10pl 1X
10mM DNTP mixture 2pl 0.2mM each
50 Mm MgCl2 3pl 1.5 Mm
Primers 100pM each 1pl each 1.0pM each
Template DNA 1-10pl 100 ng
Taq DNA Polymerase 0.5 pl 2.5 units

Autoclaved dHf)

to 100pl vol.

23

normal rate, and "4" all but one or two larvae growing at a
normal rate.

Those lines, in addition to a negative control, that
produced results of greater than or equal to 80% mortality
were noted and a second test of three to eight (line
dependent) trials using 10 larvae per leaf each was conducted.
The second replicated trial was used to confirm earlier
results and determine the lines with the highest control of
the potato tuber moth. Significance was determined by running
a Wilcoxon test utilizing SAS (Sokal and Rohlf 1995).
Morbidity scores were also taken as above to estimate the
relative health of those insects remaining alivec The
transgenic lines from the PTM bioassays were utilized later in

Southern and northern blots.

Southern Blot:

The plants that had greater than 80% potato tuber moth
mortality in the preliminary bioassays were further analyzed
by Southern blots for gene integration.

DNA Extraction protocol for Southern blot

DNA was extracted according to Saghai-Maroof et al. 1984,
modified by using 2% beta-mercaptoethanol. The DNA recovered
was quantified utilizing a Hoeffer Scientific mini-fluorometer
model TKOlOO (Hoeffer Scientific, San Fernando, California).

using a mini-fluorometer.

24
Southern blot
DNA (8-12 pg) was digested with Bam HI and run on a 1%
agarose gel at 25 volts overnighta The DNA.was immobilized to
a nylon membrane and probed with a digoxygenin-labeled Cry-V

probe labeled according to Boehringer Mannheim (Germany).

Northern Blot:

Those plants subjected to Southern analysis were also
analyzed by northern blotting. Qiagen RNeasy Plant Total RNA
Kit (Chatsworth, California) was used for all RNA isolations.
RNA (20 ug) was run on a formaldehyde/agarose gel and RNA was
immobilized to a Hybond-N nylon membrane (Amersham Life
Science, Buckinghamshire, England) and probed with a
dioxygenin-labeled Cry-V probe according to Boehringer

Mannheim (Germany).

RESULTS

Plant Transformation with CryV-Bt:

Putative transgenic shoots were produced on Lemhi Russet leaf
explants as quickly as four weeks and continued for two
months. A total of 60 individual shoots were removed and
transferred.to kanamyacin rooting medium.(50‘mg/l). The first
33 lines to root in this medium were then assayed to determine
the presence of the CryV-Bt gene. A total of 27 of these 33
lines were PCR positive for the CryV-Bt via PCR. Many non-
transgenic plants developed roots, but these roots only grew
on the medium surface and up the side of the culture tube. In
contrast, putative transgenics produced roots that grew into
the medium to the bottom of the culture tube.

Line USDA8380-1 was slower in shoot regeneration, with.an
average of eight to twelve weeks required to obtain shoot
production. However, once shoots were obtained they grew
rapidly in kanamyacin rooting medium. Initially, 60 shoots
were selected of which 30 rooted. Twenty-six of 27 rooted
plants were PCR positive for CryV-Bt.

L235-4 was the most.difficult.to obtain transgenic lines.
It took three times as many leaf disks as Lemhi Russet to
obtain 45 healthy shoots for the rooting assay. Shoot

regeneration occurred between 8-12 weeks. Only one-third of

25

26
the shoots rooted in the kanamyacin rooting medium, of which

14 were PCR positive for the CryV-Bt gene.

Potato tuber moth bioassays:

A total of 18 PCR-positive Lemhi Russet CryV-Bt lines
(Figure 1) and non-transgenic Lemhi Russet were used for the
preliminary potato tuber moth bioassays. Average potato tuber
moth mortality ranged from 20% to 95% mortality. All Lemhi
Russet transgenic lines had a higher mortality level than the
non-transgenic Lemhi Russet (Table 6). The eight lines that
demonstrated a mortality of 80% or greater were then used for
further potato tuber moth mortality and morbidity studies
(Table 7).

Twenty-five 'transgenic lines. of 8380-1 CryV-Bt. were
tested in a preliminary feeding trial. 8380-1 demonstrated a
high level of variability in mortality between trials which
increased the difficulty of identifying the transgenic lines
with the greatest host plant resistance. The non-transgenic
8380-1 had 50% potato tuber moth mortality in the initial
bioassay, while the CryV-Bt 8380-1 lines ranged from 40 to
100% (Table 8).

Eleven lines that demonstrated 90% or higher mortality
were advanced to a second bioassay (Table 9). In this second
bioassay, ten insects per leaf were used with.two replications
to provide enough differentiation to select the eight best

lines for further testing. Mortality for 8380-1 was 60%,

27

 

Figure 1. CryV-Bt PCR Results of Lemhi Russet. Lanes are 1-20
In lane one is Lambda restriction enzyme cut

left to right.
with BStE II. Lanes 2—17 have rooting assay positive Lemhi
Lane 18 has Plasmid DNA containing the CryV-Bt gene.

lines.
Lanes 19 and 20 are non-transgenic lines.

28

Table 6. Means of potato tuber moth preliminary tests of Lemhi
Russet transgenic lines in detached leaf bioassays.

 

 

Line Mortality
(percent)
Lemhi Russet 40
Lemhi-l 8O
Lemhi-2 80
Lemhi-4 7O
Lemhi-6 60
Lemhi-7 90
Lemhi-9 90
Lemhi-10 90
Lemhi-11 70
Lemhi-12 85
Lemhi-l3 65
Lemhi-14 80
Lemhi-15 90
Lemhi-17 95
Lemhi-18 65
Lemhi-19 20
Lemhi-20 9O
Lemhi-21 85

Lemhi-22 8O

 

29

Table 7. Potato tuber moth means of Lemhi Russet transgenic
lines in detached leaf bioassays.

 

 

Line Mortality Standard
(percent) Deviation
Lemhi Russet 3.3 4.7
Lemhi-1 90* 2.7
Lemhi-7 93* 2.7
Lemhi-12 83* 2.7
Lemhi-l4 93* 2.7
Lemhi-15 83* 2.7
Lemhi-17 88* 3.9
Lemhi-21 86* 2.7
Lemhi-22 88* 3.9

 

*Significantly different from the untransformed line, p<0.05,
Wilcoxon test.

30
while the CryV-Bt lines ranged from 70 to 90%.

These eight 8380-1 CryV-Bt lines were then tested
inbioassays with ten first instar larvae per leaf of six
replications. All transgenic lines except 8380-1.5 caused
significantly higher potato tuber moth mortality than 8380-1,
p=0.01 (Table 10).

L235-4 and eleven L235-4 CryV-Bt transgenic lines were
used in potato tuber moth feeding bioassays. A trial of two
replications with ten first instar larvae per leaf was first
conducted. From these tests eight lines were selected for
further potato tuber moth bioassays” The average potato tuber
moth.mortality ranged from 80% to 96% mortality in the CryV-Bt
transgenic lines. Three more replications with ten first
instar larvae were conducted. All transgenic lines had higher

potato tuber moth mortality than L235-4 (Table 11).

Southern and northern analysis
The lines listed in Table 12 demonstrating the highest
level of potato tuber moth resistance were used for Southern
and northern analyses. The Southern analysis confirmed the
presence of the CryV-Bt gene in all lines tested (Fig 2).
Northern analysis demonstrated that all but one CryV-Bt
line had similar transcription levels. The transgenic line
8380-1.19, which had the lowest level of potato tuber moth
resistance did correlate with a lower level of transcription

of the CryV-Bt gene (Fig 3). Significance tests were not run

31
on morbidity for any line but mean scores are listed in

Appendix A.

32

Table 8. Means for potato tuber moth mortality preliminary
trials of 8380-1 Cry-Bt lines and untransformed 8380-1 in
detached leaf bioassays.

 

 

Line Mortality
(percent)

8380-1 50
8380-1.1 90
8380-1.2 60
8380-1.4 60
8380-1.5 90
8380-1.6 100
8380-1.7 60
8380-1.8 70
8380-1.9 90
8380-1.10 70
8380-1.11 80
8380-1.12 60
8380-1.13 80
8380-1.14 90
8380-1.15 4O
8380-1.16 90
8380-1.17 80
8380-1.18 90
8380-1.19 100
8380-1.20 88
8380-1.21 40
8380-1.22 50
8380-1.23 80
8380-1.24 100
8380-1.25 100

8380-1.26 100

 

33

Table 9. Means for potato tuber moth mortality second
preliminary trials of 8380-1 Cry-Bt lines and untransformed
8380-1 in detached leaf bioassays.

 

 

Line Mortality
(percent)
8380-1 60
8380-l.l 9O
8380-l.5 9O
8380-1.6 7O
8380-l.9 7O
8380-l.14 7O
8380-1.16 8O
8380-l.18 85
8380-l.19 75
8380-1.24 70
8380-1.25 75

8380-1.26 75

 

34

Table 10. Means for potato tuber moth.mortality of 8380-1 Cry-
Bt lines and untransformed 8380-1 in.detached leaf bioassays.

 

 

Line Mortality Standard
(percent) Deviation
USDA 8380-1 54 9.1
8380-l.l 75** 9.3
8380-l.5 78* 9.3
8380-l.9 88** 9.3
8380-1.16 80** 9.4
8380-1.18 78** 8.5
8380-1.l9 69** 9.2
8380-l.25 87** 9.3
8380-1.26 78** 9.3

 

* Significantly different from untransformed line, p<0.05,
Wilcoxon test.
**Significantly different from untransformed line, p<0.01,
Wilcoxon test.

35

Table 11. Means for potato tuber moth of L235-4 CryV-Bt
transgenic lines in detached leaf bioassays.

 

 

Line Mortality Standard
(percent) Deviation
L235-4 4 8.0
L235-4.3 96** 4.6
L235-4.5 90** 4.6
L235-4.8 96** 4.5
L235-4.11 90** 4.6
L235-4.12 90** 4.6
L235-4.13 96** 4.5
L235-4.l4 88** 4.6
L235-4.l6 96** 4.5

 

**Significantly different from untransformed line, p<0.01,
Wilcoxon test.

36

 

Figure 2. Southern blot of Bam HI digested DNA from L235-4
CryV-Bt lines used in insect feeding studies. probed with
CryV-Bt gene.

37

Figure 3. Northern analysis of total RNA from 8380-1 CryV-Bt

transgenic lines probed with CryV-Bt gene fused with the GUS
gene.

 

38

Table 12. Lines used for Southern and northern analysis.

 

8380-1 Lemhi Russet L235-4
8380-1.l Lemhi-l L235-4.3
8380-l.5 Lemhi-7 L235-4.5
8380-l.9 Lemhi-12 L235-4.8
8380-l.l6 Lemhi-l4 L235-4.ll
8380-1.18 Lemhi-15 L235-4.12
8380-1.19 Lemhi-l7 L235-4.13
8380-1.25 Lemhi-21 L235-4.14

8380-1.26 Lemhi-22 L235-4.16

 

DISCUSSION
The purpose of this study was to combine natural host plant
resistance mechanisms with the CryV-Bt gene, via
transformation to confer host plant resistance to potato tuber
moth. The Agrobacterium-based transformation protocol proved
highly efficient with 165 total shoots recovered from three
genotypes. Not all rooted regenerates were tested, but 67 of
75 were confirmed PCR positive. From the PCR positive lines,
five lines with 80% or greater potato tuber moth mortality
were obtained for both Lemhi Russet and L235-4. The
transformation/regeneration protocol of Yadav and Sticklen
(1995) used in this research produced transgenic lines for
threejpotato»genotypes (two with wild.Solanum spp. background)
suggesting broad genotypic adaptability of this protocol.
Compared with previous transformation experiments, this was
highly successful. Sheerman and Beven (1988) experienced
highly genotype-dependent responses and in some cases did not
recover any transgenic lines for some genotypes“ The De Block
(1988) protocol has produced mixed results in our laboratory;
producing many regenerates and transformants for some
cultivars and minimal to none for others (data not shown).
Stiekema (1988) utilized 'tuber ‘tissue for .Agrobacterium-
mediated transformation with only 1% of recovered shoots
transgenic. Newell et al. (1991) proposed a transformation
technique using potato stem sections. There was a transgenic

recovery of 2-5% which is considerably lower the 30-50% level

39

40
obtained herein.

The CryV-Bt differs from the other Bt constructs in that
its specificity is for Coleoptera and Lepidoptera insects.
CryI-Bt is Lepidoptera specific, CryII-Bt is specific to
Lepidoptera and Dipteran, CryIII-Bt is Coleoptera specific,
and CryIV-Bt is Dipteran specific (Van Rie et al. 1994).
Codon modification has been shown to increase gene expression
(Perlak 1990). The wild type Cry I was shown to have little
or no effect (Ebora et al. 1994). However, mortalities as
high as 80% were recorded once codon modification was carried
out in CryIA (Wunn et al. 1996). The potato tuber moth
mortalities using our CryV-Bt transgenics were as high or
higher than those observed for the codon-modified CryIA (Wunn
et al. 1996). An additional value of this particular
construct is that it is potentially active against two of the
major pest orders in potato. Control with the CryV-Bt gene is
an order of magnitude lower for control of Colorado potato
beetle (Leptinotarsa decemlineata) than Cry III-Bt (Jan
Tippet, ICI Seeds personal communication).

Examining Bt efficacy in the 8380-1 CryV-Bt lines is
problematic. The leaf bioassay cannot distinguish between Bt
and leptine effects. Leptines in the leaves of 8380-1 caused
70-100% mortality of Potato tuber moth within 72 h (walter
Pett, personal communication). These levels of mortality were
also typical of the mortalities with Lemhi Russet transgenic

lines only containing CryV-Bt. Difficulty was encountered in

41

maintaining healthy detached leaves of 8380-1 for 72 h,
because the leaves either wilted or callused. Because of
these two situations, the potato tuber moth bioassays were
reduced to 48 h in order to determine control due to CryV-Bt
rather than the leptines. The reduction in time reduced the
mean mortality in 8380-1 due to leptines down to 53%. This
mortality level was low enough to allow the distinction
between CryV-Bt lines. When a few detached leaf assays were
continued for 72 hours usually 100% insect mortality was
observed (data not shown).

To maintain uniformity of testing, all transgenic lines
were tested using the 48 h testing period. Lemhi Russet and
L235-4 showed good control. L235-4 CryV-Bt lines, with 88%-
96% potato tuber moth mortalities, were the highest levels of
CryV-Bt control observed in this study. The Lemhi Russet
CryV-Bt lines demonstrated a range of 83%-93% potato tuber
moth mortalities. The lowest mortalities observed in this
bioassay were 8380-1 CryV-Bt lines ranging from 69%-88% potato
tuber moth mortalities.

The construct used had the B-glucuronidase (GUS) gene
attached to the CryV-Bt gene, with the 358 promoter activating
both genes. This research demonstrates that high levels of
expression of the CryV-Bt can be obtained, and northern blots
indicated that the GUS gene was being transcribed with the Bt
gene. It has been observed that the GUS gene may interfere

with gene expression in potato (Belknap et al. 1994). Future

42
constructs to be 'tested. will compare transgenic CryV-Bt
constructs with and without the GUS gene.

Some somaclonal variation was observed in the phenotypes
of any of the transgenic lines. There was some minor
distortion of leaf morphology observed in two of the low
expressing Lemhi Russet transgenic lines that were not
selected for further investigation. Lemhi Russet and L235-4
also produced plants with no apical dominance. These lines
never left tissue culture.

Further investigation into CryV-Bt expression and leptine
production in the transgenic lines would be useful.
Biochemical characterization should be done to determine Bt
and leptine levelsu ‘Western blot analysis would.determine the
translation of the CryV-Bt to determine the Bt protein levels
111 the transgenic lines. It. was assumed. that leptine
production in the transgenic plants is the same as non-
transgenic 8380-1, but gas-liquid chromatography (GLC) for
leptine content should be conducted for confirmation (Sinden
1986).

The potato tuber moth detached leaf bioassay involved an
interaction of three factors: 1) the health of the potato
tuber moth, 2) maturity and health of the plant, and 3)
environmental conditions in which the bioassays were
conducted. These factors could influence the potato tuber
moth mortality levels observed. For example, the preliminary

leaf bioassay’ for' Lemhi Russet. was conducted.‘under' high

43
temperature and low light conditions. Once the location was
changed to improve environmental conditions, Lemhi Russet leaf
quality improved. In addition, a 5% increase in potato tuber
moth mortality was observed in transgenic lines and a 10%
reduction in mortality on the control lines.

The health of the potato tuber moth affected the tests
most notably from the standpoint of larvae number availability
for bioassays. The initial room conditions in which the
potato tuber moth were reared varied greatly in temperature
over time, with excessively high temperatures making a
significant impact on population size especially during the
summer months. High temperature affected both the number of
eggs laid and the larvae hatching from ‘those eggs. .A
relocation of the potato tuber moth cultures to a growth
chamber with optimal constant temperature (26TH and humidity
(99%), egg and larvae numbers increased two-fold and five-
fold, respectively (Chavez et al. 1988). Greater larvae
numbers allowed for more genotypes to be tested at a time as
well as optimum larval number per leaf. A reduction in insect
health may have contributed to higher potato tuber moth
mortality under the unstable environmental conditions,
however, not enough to effectively ascertain to what degree.

In this research, the larvae number was a major limiting
factor to the preliminary leaf bioassays. Five first instar
larvae were used for 8380-1 and Lemhi Russet preliminary leaf

bioassays. By the time L235-4 CryV-Bt lines were tested, the

44

potato tuber moth population had rebounded.to a level to allow
for ten first instar larvae to be used. In addition, there
were fewer L235-4 CryV-Bt transgenic lines to be tested.

The results from the CryV-Bt transgenic lines of Lemhi
Russet and L235-4 demonstrated that high levels of CryV-Bt
expression can be achieved with this gene construct. If Bt
expression is to be used for host plant resistance management
strategies, then the next step is to look at how the lines can
be used in a potato tuber moth resistance management strategy.
Host plant recognition by phytophagous insects occurs in five
stages: 1) host-habitat finding, 2) host finding, 3) host
recognition, 4) host acceptance, and 5) host suitability
(McGaughey & Whalon 1992). If the process is disrupted, then
the host is not accepted (Kogan 1982). The CryV-Bt gene
affects the recognition process at the host suitability stage,
by binding to the midgut membrane (Parenti et al. 1995). Both
the trichomes and leptines affect, the potato tuber moth at
the host acceptance stage (Yencho 1994, Sinden 1986). Under
field conditions, the natural host plant mechanisms will
reduce the number of potato tuber moth accepting the plant as
a host and thereby reducing the number of insects being
selected for CryV-Bt resistance. Reduction in Bt selection
pressure on the potato tuber moth will reduce selection
intensity for resistance genes in the potato tuber moth
population. If some potato tuber moth mortality is produced

by the Type B glandular trichomes then the number of potato

45

tuber moth on a potato plant is reduced, hence slowing the
insect adaptation to Bt. The potato tuber moth mortality
levels in the transgenic CryV-Bt 8380-1 lines may have been
due to a reduction in either leptine or CryV-Bt protein
production. The reduction in leptines, protein, or
combination there of may interfere with the deterrent nature
of the host plant or the effectiveness of the CryV-Bt gene.
Van Rie et al. (1994) demonstrated that the Bt toxin binds to
the midgut of affected insects. This binding of the Bt gene
might interfere with the absobtion and/or metabolism of the
leptines. This would also account for the lower CryV-Bt
expression in transgenic 8380-1 lines.

The leaf bioassay utilized for these studies and high
levels of expression in the leaf were demonstrated, however
the tuber is the economically damaged organ. The next key
question to be asked is whether the potato tuber moth is
controlled by CryV-Bt in the tuber. Further investigation
would also include tuber bioassay and field studies. The
constitutive expression promoter, Cauliflower Mosaic Virus 358
promoter (CaMV 358) was used in the CryV-Bt construct (Benfey,
1990). Bioassays to determine if expression in the tuber
correlates with that of the leaf potato tuber moth expression
would be of value. Some investigation into tuber and leaf
mortality correlation studies'have beenlcompleted, but further
research is needed (Walter Pett, personal communication).

Future work will include testing of the CryV-Bt gene in

46

combination with the Gelvin super promoter (Narasimhulu.et al.
1996) and the patatin promoter (Wenzler et al. 1989) for
expression levels.

Field studies are needed to determine the value of host
plant resistance in the CryV-Bt genotypes. The leptine
resistance level can be tested via leaf assays because it is
a chemical mechanism. Trichomes are a mechanical mechanism
that affects host selection which is eliminated once an insect
is manually placed on a leaf (Kogan 1982). Thus the need for

field testing.

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52

APPENDIX A

Table 13. Summary of morbidity scores for Lemhi Russet,
L235-4, and USDA 8380-l.

 

 

Line Mean Morbidity
USDA 8380 2.1
8380-1.1 1.0
8380-1.5 0.7
8380-1.9 0.8
8380-1.16 0.8
8380-1.18 1.0
8380-1.19 1.2
8380-1.25 0.5
8380-1.26 0.8
Lemhi Russet 5.0
Lemhi-1 0.7
Lemhi-7 1.0
Lemhi-12 1.0
Lemhi-14 0.7
Lemhi-15 1.3
Lemhi-17 0.6
Lemhi-21 1.0
Lemhi-22 0.8
L245-4 5.0
L235-4.3 0.5
L235-4.5 0.6
L235-4.8 0.4
L235-4.11 0.8
L235-4.12 0.6
L235-4.l3 0.4
L235-4.14 1.0
L235-4.16 0.4

 

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