 

{77/4579

LIBRARY
Michigan State
University

This is to certify that the
thesis entitled !

POTATO TUBERWORM (LEPIDOPTERA:
GELICHIIDAE) RESISTANCE IN POTATO LINES WITH
THE Bacillus thuringiensis-cry1ac GENE AND
NATURAL RESISTANCE FACTORS

presented by
MARIA ALLASAS ESTRADA 1

has been accepted towards fulﬁllment
of the requirements for the

MS. degree in Plant Breeding and Genetics

 

Ewﬂ X>W

Major Professor’s'Signature

August 15, 2005

 

Date

MS U is an Affirmative Action/Equal Opportunity Institution

..—.-n-.-.—v—

 

 

PLACE IN RETURN BOX to remove this chedcout from your record.
TO AVOID FINES retum on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE

DATE DUE

DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2105 cJCIIRCIDatooqundd-pds

 

POTATO TUBERWORM (LEPIDOPT ERA: GELICHIIDAE) RESISTANCE IN
POTATO LINES WITH THE Bacillus thuringiensis-crylac GENE AND
NATURAL RESISTANCE FACTORS
By

Maria Allasas Estrada

A THESIS
Submitted to
Michigan State University

in partial fulﬁllment of the requirements
for the degree of

Master of Science

Plant Breeding and Genetics Program
Department of Crop and Soil Sciences

2005

ABSTRACT
POTATO TUBERWORM (LEPIDOPTERA: GELICHIIDAE) RESISTANCE IN
POTATO LINES WITH THE Bacillus thuringiensis—crylac GENE AND
NATURAL RESISTANCE FACTORS
By
Maria Allasas Estrada

The potato tuberworm (Phthorimaea operculella (Zeller)) is one of the most
common and destructive insect pests to potato (Solanum tuberosum L.). Previous host
plant resistance work has not produced potato material that has appreciable levels of
resistance to potato tuberworm. A potential solution to improve cultivated potato
resistance to potato tuberworm is through combining resistance mechanisms of
glycoalkaloids and glandular trichomes with cry genes. The objectives of this study were:
1) transform three potato lines which differ in natural host plant resistance with a codon-
modiﬁed Bt-crjylac gene; 2) verify insertion of Bt-cry lac, quantify the protein
expression, and determine the number of inserted copies of the gene; and 3) conduct
laboratory bioassays to test the potato tuberworm response on Bt— cry lac transgenic lines.
Putative transgenic lines of Spunta, ND5873-15 & NY123, were developed using vector
construct (pSPUD 15) with the codon-modiﬁed Bt-cryl ac gene. Integration of Bt—cryl ac
gene in Spunta and ND5873-15 transgenic lines was conﬁrmed by the PCR and Southern
blot analysis. Protein expression in the transgenic lines (0-0.58 ng/mg) was conﬁrmed by
ELISA. Detached leaf bioassays with neonate larvae of potato tuberworrn showed that
crylac gene was effective in controlling potato tuberworm 1St instar larvae (up to 97 %
mortality). Combining natural host plant resistance and engineered resistance with the
Bacillus thuringiensis-crylac gene in potato plant was effective in conferring increased

resistance to potato tuberworm.

I would like to dedicate this work to my loving husband Dexter, my little angel John
Benedict, to my family in the Philippines Tita Belle, Tito Boyet, Tatay JR & Ate Imee,
and in memory of my mother Edna.

iii

ACKNOWLEDGEMENT

Finally, after all the sacriﬁces, this manuscript becomes a reality. My sincerest
thanks to my adviser, Dr. Douchcs and to my committee members, Dr. Graﬁus and Dr.
Sink for painstakingly reading and correcting my manuscript over and over again.

I would like to thank Dr. John Kemp of New Mexico State University for letting
me use their Bt-crylac gene. Special thanks to Kelly Zarka for the vector construct and
to the staff and graduate students in potato lab, Susannah, Anne, Lynn, Jarred, Joe, Jay,
Jenna, Julie and Liz. Thanks also to the staff of Entomology lab (Adam, Walt &
Franciso), Sugar beet lab (Scott and Suba), Dr. Sticklen’s lab (Dr. Sticklen and Callista),
and Bean lab (Dr. Kelly, Halima & Anne).

Much thanks to Tita Marilyn & Grandma for always lending a helping hand; and
to Mama Edith for taking care of my little JB.

To Hesham, thank you very much for helping me in the Southern blot analysis.

To Mary Gebbia, Rey Ebora and Rita House thank you so much.

Lastly, thanks to IIE for giving me the chance to ﬁnish my MS through a
Fulbright Scholarship Grant.

To all of you, my indebt appreciation is beyond words ........

iv

TABLE OF CONTENTS

LIST OF TABLES ............................................................................. vi
LIST OF FIGURES ........................................................................... vii
INTRODUCTION ............................................................................. 1
Potato ................................................................................. 1
Potato Tuberworm .................................................................. 2
Host Plant Resistance ............................................................... 2
Genetic Engineering ................................................................. 5
Bacillus thuringiensis ............................................................... 6
OBJECTIVES ................................................................................. 8
MATERIALS AND METHODS .......................................................... 9
Vector Construct .................................................................... 9
Plant Materials ....................................................................... 9
Transformation Protocol ........................................................... 9
Potato tuberworm rearing ........................................................... 12
Detached-leaf Bioassay ............................................................. 12
Tuber Bioassay ...................................................................... 13
Molecular Characterization ........................................................ 13
Statistical Analysis .................................................................. 17
RESULTS ...................................................................................... 18
Potato Transformation with Bt-Cryl ac gene .................................... 18
Molecular Characterization ........................................................ 18
Detached-leaf Bioassays ........................................................... 24
Tuber Bioassay ...................................................................... 25
DISCUSSION ................................................................................. 31
APPENDIX A ................................................................................ 37
APPENDIX B ................................................................................ 38
LITERATURE CITED ...................................................................... 39

LIST OF TABLES

Table 1. Results of Kanamycin rooting assay and PCR ampliﬁcation of putative Spunta

and ND5873-15 transgenics .................................................................. 19
Table 2. Mean protein expression of Spunta and ND5873-15 transgenic lines 23
Table 3. DNA concentrations used in the PCR reaction ................................... 37

Table 4. Total glycoalkaloids contents of ND5873-15, ND5873-15 transgenic lines,
Spunta and Spunta transgenic lines ............................................................ 38

vi

LIST OF FIGURES

Fig. 1. Schematic diagram of pSPUD 15 vector construct. RB, T-DNA right border;
NOS-T; CryIa(c), Bt cry] ac insecticidal protein gene; CaMV 35S Pro, promoter; NOS-T;
NPTII, neomycin phosphotransferase gene confers resistance to kanamycin; NOS-Pro;
LB, T-DNA left border ........................................................................ 10

Fig. 2. PCR analysis of putative transgenic lines of Spunta and ND5873-15 .......... 20

Fig. 3. Southern analysis of ND5873-15 and Spunta crylac transgenic lines. DNA was
digested using XbaI and probed with cryIAc gene ......................................... 22

Fig. 4. Mean percent mortality (detached-leaf bioassay) of ﬁrst instar of potato
tuberworm on Spunta and Spunta transgenics .............................................. 27

Fig. 5. Mean percent mortality (detached-leaf bioassay) of ﬁrst instar of potato
tuberworm on the ND5873-15 and ND5873-15 transgenics .............................. 28

Fig. 6. Mean percent mortality (5-day detached-leaf bioassay) of ﬁrst instar of potato
tuberworm on the control ND5873-15, ND15.1O and ND15.11 ......................... 29

Fig. 7. Mean percent mortality (tuber bioassay) of ﬁrst instar of potato tuberworm on the
ND5873-15 and ND5873-15 transgenic lines .............................................. 30

vii

INTRODUCTION

POTATO

Potato (Solanum tuberosum L.) is one of the most important crops for human
nutrition worldwide and is a healthy source of I carbohydrates, high quality protein,
essential vitamins, minerals, and trace elements (Flanders et a1. 1999). It is the highest
ranking vegetable crop in production in the USA (Douches et al. 1996). Estimated
production is ca. 300 million tons per year (Gebhardt et al. 2001 ). The three main targets
of potato breeding are for ﬁesh food market, processing industry, and for non-food
industrial uses. At present, potato breeders are giving more emphasis in meeting both the
quality requirements of processors and supermarkets, and the yield potential to ensure the
commercial success of the variety they are releasing. In addition, they are also focusing
their breeding efforts toward developing varieties that are resistant to insect pests and
diseases. Potato is vegetatively or clonally propagated crop and is therefore vulnerable to
many diseases and insect pests that affect both the foliage and the tuber (Gebhardt et a1.
2001). Furthermore, potato has numerous pest and pathogens that can reduce yields and
overall plant vigor (Coombs et al. 2001).

The present trend in potato breeding is combining quality with resistance to insect
pests and diseases. Priorities are now being given toward resistance breeding to potato
cyst nematode, late blight, blackleg and powdery scab, blemish diseases, storage diseases,
viruses, and some of the most common destructive and most important insect pests:

Colorado potato beetle, potato tuberworm and European corn borer (N aimov et al. 2003).

POTATO TUBERWORM

One of the most common and destructive insect pests to potato worldwide, both in
tropical and subtropical areas, is the potato tuberworm (Phthorimaea operculella
(Zeller)) (Flanders et al. 1999). Potato tuberworm causes damage in both ﬁeld and
storage (Westedt et al. 1998), reduces the quality of produce, and increases the risk of
pathogen infection. Furthermore, damage caused by potato tuberworm can tremendously
reduce the potato yield because it attacks both the foliage and the tuber (Capinera 2001).
In warmer climates, the quantity and quality losses in storage can be as high as 100%
(Lagnaoui et al. 2000). Potato tuberworm life cycle, feeding habit and its ability to
develop resistance to chemical insecticides is an increasing agricultural problem in the
tropical and subtropical areas in the world (Naimov et al. 2003). Potato tuberworm spend
their larval stages either in the foliage or in the tuber. The larvae mine both the foliage
and the petiole creating transparent leaf blisters. Damage in the foliage caused by potato
tuberworm may be sufﬁcient to cause severe damage or death to the plant. The eggs that
were oviposited on the exposed potato tubers will hatch and will then enter through the
"eyes". They will make slender tunnels throughout the tuber and introduce bacterial rots.

Mounds of frass at the tunnel entrances can give indication of an infested tuber.

HOST PLANT RESISTANCE

Varying levels of resistance to insects occur naturally in crop plants and closely
related species (Stoner 1996). Insect resistance appears to be a primitive trait in wild
potatoes (Flanders et al. 1999).

Solanum spp. exhibit a wide variety of defense mechanisms against insect pests.

The two general types of host plant resistance that can be found in Solanum are high

levels of glycoalkaloids and glandular trichomes. Potatoes contain glycoalkaloids that
have long been known to possess antimicrobial and insecticidal properties (Tingey 1984).
The glycoalkaloids present in the leaves can also act as insecticidal compounds. The two
most common glycoalkaloids found in potatoes are a-chaconine and a-solanine, together
they comprise as much as 95% of the total glycoalkaloid present in the potato (Lachman
et al. 2001). Chemically, ct-chaconine and a-solanine, are classiﬁed as steroidal
glycoalkaloids. Solanum steroidal glycoalkaloids are large biologically-active secondary
metabolites that have been isolated from more than 350 plant species (Lawson et al.
1993, Roddick 1986, Ripperger & Schreiber 1981). These are toxins that occur naturally
in many edible and non-edible members of the Solanaceae, such as eggplant (Solanum
melongena) and potato (Solanum tuberosum). Presence of steroidal glycoalkaloids in
potato could both be a beneﬁt and a concern (Lawson et al. 1993). Consumption of tubers
with high steroidal glycoalkaloids concentration can be detrimental to the health of
human and animals. The Solanum glycoalkaloids are cholinesterase inhibitors which
result in neural function impairment (Hopkins 1995, Lachman et al. 2001). The fatal oral
dose for an adult would be 420 mg (Lachman et al. 2001). Glycoalkaloids concentration
in potato plants can be highly affected by the temperature. This explains why these
protective glycoalkaloids are present at higher concentration in the aerial ( leaves, stems
and sprouts) part of the potato plant and are normally present in lower concentration in
the tubers (Lachman et al. 2001). Similarly, Laﬁa and Lorenzen (2000) observed a
signiﬁcant increase in foliar glycoalkaloids when plants were grown at higher
temperature (32/27 °C).

Potato possesses another type of defense mechanism classiﬁed as glandular

trichomes. Wild species of potato have glandular trichomes that confer resistance to

different kinds of insect pests. The most common types of glandular trichomes that can
be found on the potato foliage are the type A and type B. A recently discovered wild
potato species, S. neocardenasii Hawkes & Hjerting (series Tuberosa), from central
Bolivia were observed to has glandular hairs in the foliage (Lapointe et al. 1986, Hawkes
et al. 1983). This Bolivian wild potato is resistant to numerous insect pests like the potato
tuberworm complex. Type A and type B glandular trichomes are associated with insect
resistance in S. berthaultii (Yencho et al. 1994, Tingey 1991). Resistance of S.
berthaultii to insects is associated with chemical factors localized in the glandular
trichomes, in addition, a feeding deterrent is present in the trichomes (Tingey et al. 1994).
Previous studies of droplets on the B trichomes showed that the inheritance of this
character is inherited as a single dominant gene (Kalazich 1991, Gibson 1979).

Although, potato has natural resistance to insect pests, intensive agricultural
practices created an artiﬁcial environment that favors insect pest proliferation. Today, a
tremendous increase in the use of chemicals to control insect pests has created a strong
pressure for the development of resistant insect populations and reduced insect predators’
populations. Because of this, current efforts to develop insect-resistant crops are
following the theme of transforming plants with a single gene encoding insecticidal
enzymes or toxins. The delta endotoxins of Bacillus thuringiensis are the most widely
researched genes among the insecticidal enzymes or toxins (Barton and Miller 1994).

One of the key components of an integrated pest management program to control
potato tuberworm is host plant resistance. At present, there is no potato material,
produced from host plant resistance breeding that has appreciable levels of resistance to
potato tuberworm (Lagnaoui et al. 2000). Combining natural host plant resistance and

insect resistance conferred by a Bacillus thuringiensis gene in potato breeding against

potato tuberworm may increase the efﬁcacy and stability of resistance (Coombs et al.

2002)

GENETIC ENGINEERING

A potential solution for the improvement of cultivated potato for traits such as
resistance to diseases, insect pests, herbicides, environmental stresses, yield and as well
as various quality parameters is through the use of genetic engineering (Conner et al.
1994). One highly successful approach to engineering resistance has involved generating
plants that synthesize antimicrobial or insecticidal products (Dempsey et al. 1998). In
terms of genetically engineering potato for host plant resistance to insects, there are two
classes of important toxin genes: those coding for toxins against the Colorado potato
beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera:Chrysomelidae), and those
coding for toxins active against lepidopterous pests, such as potato tuberworm (PTW),
Phthorimaea operculella (Zeller) (LepidopterazGelechiidae) (Gould et al. 1994).

After the discovery of a successful transformation procedure using the vector
Agrobacterium tumefaciens, potato has been used as a subject for genetic engineering
experiments for the past 20 years. Agrobacterium-mediated gene transfer has been used
as one of the most common transformation techniques in potato breeding (Conner et al.
1994). Introduction of transgenes into higher plants using Agrobacterium—mediated
transformation method still remains as the predominant protocol used for potato (Belknap
et al. 1994). This procedure offers the advantage of mobilizing deﬁned regions of DNA

into the nuclear genomes of the plant (Belknap et al. 1994).

Bacillus thuringiensis

Naturally occurring Bt strains possess genes which code for a number of distinct
toxic proteins (ES-endotoxins) with different spectra of activity against insects (Gould et
al.1994). Potato has been genetically transformed to express genes of various subspecies
of Bacillus thuringiensis Berliner (Bt) encoding insecticidal protein (Head et al. 2002).
For the past ten years, 5-endotoxins from different Bacillus thuringiensis (Bt) subspecies
have been used to protect crops against insects (Dempsey et al. 1998). Transformation in
potato using Bt-cry genes have advantages. The mode of action of these genes is highly
speciﬁc or is highly host speciﬁc. A protein with speciﬁc toxicity towards coleoptera
would not be toxic to other orders of insects, and Bt crystal proteins have not shown any
toxicity towards humans, other mammals or birds (Coombs et al. 2002, Lavrik et al.
1995). Furthermore, transgenic plants deliver the Bt toxin gene with increased efﬁcacy
compared to foliar application, lowering application costs (Douches et al. 2001). At
present, over 10 million hectares are planted to Bt crops globally, mainly with plants
expressing toxins against lepidopteran pests, butterﬂies and moths (Ferry et al. 2004)

Bt produces different crystal (Cry) insecticidal proteins during sporulation, which
provide protection against a wide range of insect pests (Jenkins and Dean 2001). The
crystal protein, when ingested, is solubilized in the insect’s midgut and releases 6-
endotoxins upon activation by the insect’s midgut proteases. The activated toxins bind to
the midgut epithelium of the insect causing disruption in membrane integrity leading to
rapid death (Gill et al. 1992). The insecticidal crystal protein produced by Bacillus
thuringiensis var. kurstaki is a toxin speciﬁc for Lepidoptera larvae (Chan et al. 1996).
Among the ﬁrst genes that have been used in genetic transformation of plants for

improved insect resistance were genes encoding Bt crystal proteins (Theunis et al. 1998).

Bt toxin genes have been cloned, sequenced and codon-modiﬁed to increase
expression level in plants and are now being used in various crop species (Mohammed et
al. 2000). Potatoes can be transformed using Agrobacterium tumefaciens Ti plasmid-
_ mediated genetic transformation (Douches ct. al. 1998). When inserted into crop plants,
the efﬁcacy of wild type Bt genes is less than that of a codon-modiﬁed Bt-cryl and Bt-
cry3 genes (Douches et. al. 1998). Transformation with wild type cry lac Bt toxin gene
speciﬁc for Lepidoptera have produced potatoes with low levels of Bt-expression causing
from 20-60% insect mortality in detached leaf bioassays (Mohammed et. al. 2000). The
use of a codon-modiﬁed Bt gene could provide increased receptor binding and toxicity,
broadening the spectrum of activity against target pests, and reducing effects on non-

target insects (Jerkins and Dean 2001).

Objectives of the study

1) transform three potato lines which differ in natural host plant resistance
(susceptible, glandular trichomes, increased glycoalkaloids) with a codon-
modiﬁed Bt-cryl ac gene;

2) conduct laboratory bioassays to test the potato tuberworm response on Bt- cry
Iac transgenic lines and;

3) conduct molecular characterization of the Bt-cry 1 ac transgenic lines to verify
gene insertion, to quantify the amount of protein and to determine the number of

inserted copies of the gene.

MATERIALS AND METHODS

VECTOR CONSTRUCT

pSP 73 cryla(c) improved [from John Kemp, New Mexico State University] was
digested with X7201 and T4 DNA Polymerase treated to create a blunt end. It was then
digested with BamHI and the 1.76 Kb fragment was isolated. This fragment was ligated
to the 11 Kb fragment of pBIlZl which was cut with BamHI and EcoICRI. The resulting
plasmid is the pSPUDlS (Fig.1). The plasmid includes the following: RB, T-DNA right
border; NOS-T; cryla(c), Bt cry1a(c) insecticidal protein gene; CaMV 35$, promoter;
NOS-T; nptII, neomycin phosphotransferase gene confers resistance to kanamycin; NOS-

Pro; LB, T-DNA left border.

PLANT MATERIAL

Three potato varieties were used for Agrobacterium-mediated transformation
experiment: Spunta, ND5873-15, and NY123. Spunta is a long, white ﬂeshed, tablestock
cultivar that was bred in Netherlands. It is grown widely in subtropical regions such as
North Africa and South America (Douches et. a1 2002). NY123 is a breeding line from
Cornell University that has higher densities of type A glandular trichomes. ND5873-15 is
a breeding line from North Dakota State University (NDSU) that has higher levels of an

undeﬁned glycoalkaloid in the foliage.

TRANSFORMATION PROTOCOL
A. tumefacians-mediated transformation as described by Douches et al. (1998)

and Step I and Step II regeneration media (Yadav and Sticklen 1995) were used in the

3.2.3.. to. <75? .ma— mesmmcz ”593353— 3 «2.838.. 28:3 one“ omauoumeabeaamean 592:3: 5 5&2 m Two Z 222:9...

.mmm >Ea0 "meow 589:. 323.82: .93.?» 5 63.58 GamOz 989:5 Eu: Sand. .my— 35.533 .833 3 95m: be 5.23:. ouaioaum A 95”..”—

Cmm ”new 5 uni

 

HIEem Ben

 

 

 

 

 

 

8H vegan: 8m mmm SE C :5: can
mm .82 83... >26 .82 :22 .82 as

10

generation of the transgenic lines of Spunta, NY123 and ND5873-15. Potato lines were
prepared for transformation by removing the tip and the petiole ends from tissue culture
plantlet leaves. The 25-30 leaves were then placed top-surface down on the solid Step I
media (MS salts, 3 % sucrose, 0.9 mgl’1 thiamine-HCI, 0.5 mgl'1 trans-zeatin n'boside, 2
mgl'l 2,4-D, 7 gl'l Bactoagar, pH 5.7) and precultured for 2-4 (1 (Yadav and Sticklen
1995). After 2 d, the precultured potato leaves were soaked in the log-phase A.
tumefaciens suspension for 5-10 min. The soaked leaves were blotted brieﬂy on sterile
paper towels to remove excess liquid and then they were transferred to ﬂesh solidiﬁed
Step I media for 4 (1. After 4 d, the leaves were washed using of 30 ml of sterile double
distilled water with 30p] Timentin. The leaves were blotted brieﬂy on sterile paper towels
to remove excess liquid and then they were transferred to solid Step 11 media containing
50 mgr" kanamycin and 200 mg 1'1 Timentin (Smith Kline Beecham, Philadelphia, PA)
in 10 x 100 mm Petri dishes. Leaves were transferred every 7-10 days to fresh solidiﬁed
Step 11 media.

Once the callus nodules produced 5-7 mm long shoots, shoots were excised and
placed in the rooting media (modiﬁed MS media with the addition of 50 mgl'l kanamycin
and 200 mg l'1 Timentin) in 25 x 150 mm culture tubes. One shoot per callus was
removed and transferred to the kanamycin rooting media. As soon as the shoot was
removed, the callus was then excised to prevent it from producing other shoots. This was
done to prevent production of similar transgenic lines. Timentin was added to prevent the
over-growth of the A. tumefaciens on the media.

Rooted shoots were grown in the tubes with rooting media and were propagated

in Magenta G5 vessels (5 plants/vessel). Four to ﬁve-week-old plants were transferred to

11

seedling trays (SO/tray) in the greenhouse and transplanted to 10 cm diameter pots in the

greenhouse for tissue collection, bioassays and molecular characterization.

POTATO TUBERWORM REARING

A potato tuberworm population from South Africa has been maintained at the
Department of Entomology Michigan State University since 2004. The culture was
maintained at 25°C on potato tubers following the rearing method described by
Mohammed et al. (2000). Eggs laid on the no.1 Whatman ﬁlter paper (Whatman,
Hillsboro, OR) were placed on top of sliced potato tubers in the Petri dish. Newly

hatched larvae (1 day old) were used in the detached-leaf bioassay.

DETACHED-LEAF BIOASSAY

The mortality of the potato tuberworm feeding on Bt-cryl ac transgenic lines was
tested using a detached leaf bioassay (Westedt et al. 1998). Fresh leaf was collected and
the leaf petiole was inserted in a sponge ﬁtted in a glass vial full of water. The vial was
sealed using Paraﬁlm to prevent water leakage. The leaf and the vial were placed in a
Petri dish (25 x 150 mm) with ﬁlter paper. Ten neonate larvae were placed on the surface
of each leaf. Each Petri dish was considered as a replication. Four replications per
transgenic line were used and the Petri dishes were arranged in a completely randomized
design. Counting of the live larvae and observation of their mining, growth and general
health was done under a dissecting microscope. Evaluation of larval mortality was made

and recorded 3 d aﬁer infestation.

TUBER BIOASSAY

Antibiosis was tested in the laboratory by placing tubers and potato tuberworm
larvae together in a closed containers (no-choice test) with vermiculite. Greenhouse
tubers were harvested from Spunta, NY123 and ND5873-15 lines. Each tuber was placed
inside a closed container with vermiculite and was exposed to 10 neonate larvae. Each
container was considered as a replication. Four replications per transgenic line were used
and the containers were arranged in a completely randomized design. Tests were carried
out in an insect mass rearing room under controlled temperature and humidity (25 i 2°C
and 70 :l: 5% relative humidity). After 21 d, the number of pupae developed was recorded

and the overall mortality were expressed in percentage.

MOLECULAR CHARACTERIZATION
POLYMERASE CHAIN REACTION (PCR)

Presence of the cry] ac gene in the transgenic potato lines was determined using
PCR. Isolation of total genomic DNA from greenhouse plants was done using the
DNeasy Plant Mini method (Qiagen, California). Each putative transgenic potato plant
DNA was used as a template. The cryl ac gene speciﬁc primer were used in the PCR. The
primers used for the crylAc gene were 5’ CAT GGC TAT CGA GAC CGG TTA CAC
TCC 3’ and 5’ CTG TCT ATG ATC ACA CCT GCA GTT CC 3’. The expected band
size was 1.8 Kb.

The PCR components for 20 pl reactions included 2p] of 10X buffer (Promega,

Wisconsin), 2p1 of 5.0 mM dNTP mixture, 0.6pl of 25mM MgC12(Promega, Wisconsin),

0.17111 of 3ODU & 0.13m of 3.8ODUof CryIAc primers, 3pl of templates DNA (4-

l3

lO4ng/ul), 2.5 U Taq DNA polymerase (Promega, Wisconsin), and water to a total
volume of 20p].

The PCR ampliﬁcation conditions were as follows: initial denaturation at 94°C for
5 min, 30 cycles of denaturation at 94 °C for l min, annealing at 50 °C for 1 min 30 sec,
and
primer extension at 72 °C for 1min 30 sec, and a ﬁnal extension at 72 °C for 4 min. The
reactions were held at 4 °C before the analysis. Reaction products were then
electrophoresed on a 1 % (w/v) agarose gel (Boeringer Mannheim, Indianapolis, IN)
containing ethidium bromide at 0.5 mg ml'1 in 1x Tris-acetate EDTA buffer (pH 8.0) at

80 mV for 1 h 30 min and viewed under ultraviolet light (254 nm).

SOUTHERN ANALYSIS
Plant DNA extraction

Total plant genomic DNA was extracted from the fresh leaf tissue of greenhouse-
grown tissue culture transplants using the CTAB extraction protocol (Saghai-Maroof et
al. 1984), modiﬁed by adding 2 % beta-mercaptoethanol to the extraction buffer. The
DNA was quantiﬁed using a mini-ﬂourometer following the manufacturer’s instructions
(Amersham Pharmacia Biotech, San Francisco CA, Hoeffer Scientiﬁc, San Fernando,
CA, model Hoefer DyNA® & Quant 200°).
DNA Probe Labelling

The plasmid crylac gene was isolated using Wizard® Plus SV Minipreps DNA
Puriﬁcation System (Promega, Madison, Wisconsin).

Polymerase Chain Reaction (PCR) was used to amplify the crylac gene in the

plasmid DNA. The PCR components for 20 pl reactions included 10 p1 of 2X Red Taq

l4

Ready mix (Sigma, Saint Louis, Missouri), 1 pl of SpM & 1 pl of SpM of crylAc forward
& reverse primers, lpl of template plasmid DNA 25 ng/ul, and water to a total volume of
20pL

The bands in the gel were cut off and were puriﬁed using the QlAquick Gel
Extraction kit (Qiagen, Valencia, Ca.). After gel puriﬁcation, the plasmid DNA yield was
quantiﬁed via spectrophotometer. The highest concentration of the DNA plasmid yield
was 50ng/ml and was used for the probe.

The puriﬁed plasmid DNA was used as the probe and was labelled using the DIG
High Prime DNA Labeling and Detection Starter Kit 11 following the manufacturer’s
instructions (Roche, Mannheim, Germany 2004).
DNA Digestion

DNA was digested using Xba I (Roche, Penzberg, Germany). The digestion
reaction components included 3 pl of 10x buffer, 6 pl of 10 U/ p1 Xba I, 25 pg of
genomic DNA and 1 pg of plasmid DNA, and water to a total volume of 30p]. After 3
h of digestion, the digested DNA ﬁagments were then electrophoresed in a 1.2 % (w/v)
agarose gel (Boeringer Mannheim, Indianapolis, IN).
Blot Transfer

The fragments produced in the gel were transferred to a nylon membrane
(Hybond N+, Amersham Life Sciences, Buckinghamshire, England) via Southern
blotting techniques following the manufacturer’s procedures.
Hybridization and detection of gene insertion

Hybridization was conducted overnight at 42°C in a fresh solution containing

approximately 25 ng ml’1 DIG-labeled DNA probe. Chemiluminescent detection was

15

conducted following the manufacturer’s procedures using 75 mUml'I anti-digoxigenin
alkaline-phosphatase conjugate and lml CSPD ready-to-use substrate (Roche, Manheim,
Germany). The membrane was then exposed to X-ray ﬁlm (Hyperﬁlm MP, Amersham

Life Sciences, Buckinghamshire, England) for 15-30 min and developed.

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

Pathoscreen kit from Agdia (Elkhart, IN) was used to detect the Bt-CryIAc
protein expressed in each transgenic plant. Fresh leaves were harvested from the
greenhouse. Collected leaf tissues were put in sample extraction bags. Fresh leaf tissues
(100 mg) was ground using an Agdia homogenizer attached to a 10” drill mess (Agdia,
Elkhart, IN) and extracts were diluted with PBST buffer to a ratio of 1:10 (w/v). The
enzyme conjugate (100 pl) was added per well and then 100 pl of each prepared sample
was dispensed in each test well of the ELISA plate. Also, 100 pl of positive and negative
control were dispensed in the appropriate testwells. The plate was sealed with paraﬁlm,
to prevent moisture loss, and was incubated for 2 hours. When the incubation period was
completed, the plate was washed with 1x PBST buffer 7 times. After washing, the wells
were soaked with 1x PBST buffer and allowed to sit for 1 min. After the soaking, 100 pl
of the TMB substrate solution was added in each well. The plate was set aside for 15 min
for the color to develop. Those wells that changed their color from colorless to blue were
scored positive and those wells that did not change their color or changed their color to
light blue were scored negative for presence of protein. Alter the color was developed, 50
pl of 3M of sulfuric acid was added in each well. The optical density was then measured

using the Wallac VictorTm2 V plate reader (Perkin Elmer Life Sciences, Downers Grove

IL) at 450nm.

l6

A standard curve was constructed from the controls and was used to compute the
Bt-crylAc protein expression in ng/ml. The amount of protein per milligram of fresh leaf

tissue was computed using the formula below:

Protein (ng/mg) = Protein conc. (ng/ml) x lml

 

100 mg of fresh leaf tissue

STATISTICAL ANALYSIS

Percentage mortality data for the bioassays were subjected to arcsine
(degrees[asin(sqrt(DEF/ 100))]) transformations before analysis of variance. Means were
compared using the Least Signiﬁcant Difference (LSD) in the general linear model
procedure (LSDaeoos) of SAS (SAS Inst., Inc., 2001). The transformed mortality was

converted to percentage mortality ([(sin(radians(ARCSIN)))"2]*100).

l7

RESULTS

POTATO TRANSFORMATION WITH cry] ac GENE

It took 4-7 weeks for NY123 to produce shoots from the leaf explants. A total of
23 shoots from NY123 were removed and transferred to individual tubes containing
kanamycin (50mg/l) media (Table 1). From the 23 shoots, 3 rooted and grew in the
kanamycin rooting media. Out of the 3 rooted, none tested PCR positive for cryl ac gene.
Line NY123 was the easiest to regenerate shoots but was the most resistant to
transformation.

A total of 32 putative transgenic potato shoots were produced from Spunta leaf
explants. Out of the 32 shoots, 12 rooted in the kanamycin media. These 12 shoots that
rooted were then regenerated into plants. Eleven of 12 rooted plants tested positive via
PCR for having the cry] ac gene. Spunta was slower to regenerate shoots compared to
NY123. On the average, it took 5-8 weeks for Spunta to produce shoots.

Line ND5873-15 was the slowest in shoot regeneration, it required 8-10 weeks for
ND5873-15 to produce shoots. A total of 34 putative transgenic potato shoots were
produced from the leaf explants and were transferred to individual tubes containing
kanamycin rooting media. Out of the 34 shoots, '12 rooted and were regenerated into
plants. The eight of 12 rooted shoots were conﬁrmed PCR positive for the presence of

cryl ac gene.

MOLECULAR CHARACTERIZATION
Presence of crylac gene in almost all (91.7%) of the rooted Spunta shoots and

66.7% of ND5873-15 rooted shoots was conﬁrmed positive by PCR (Figure 2). All the

18

Table 1. Results of kanamycin rooting assay and cry] ac PCR ampliﬁcation of putative

NY123, Spunta and ND5873-15 transgenics.

 

 

Potato Line Resistance No. of No. of rooted No. of my] ac
Factor Shoots Shootsa PCR positive lines

Spunta None 32 12 1 1

ND5873-15 Glycoalkaloid 34 12 8

NY123 Glandular trichome 23 3 O

 

a . . . . . .
The kanamycrn concentration used in the rooﬁng media was 50 mg per liter.

l9

 

2833 02> «.058 €2.00 30
203.2
203md-3
9.3.3
2.3.:
9.3.3
9.3.8
9.3.3
2.3.8
33.03
m13.§
2.3.8
2.3.8
2.3.2
mus—.8

_ 5. F252

 

Ewan—E 02> «dine c5230 3V
2.39.;

203.5

203.:

203.5

203.8

20:3

203.03

_ 5. banana

-15.

Figure 2. PCR analysis of putative crylac transgenic lines of Spunta and ND5873

20

crylac transgenic lines that tested PCR positive from Spunta and ND5873-15 were
subjected to ELISA test for protein expression and detached leaf bioassays. Southern
blotting and ELISA tests were used as the conﬁrmatory analysis for the cryl ac gene
insertion and Cry] ac protein expression. The four high performing transgenic lines from
ND5873-15 and transgenic lines from Spunta identiﬁed from the detached-leaf bioassay
were further characterized using the Southern blotting analysis.

Cry] ac protein expressions in Spunta and ND5873-15 transgenics varied between
0 to 0.58 ng/mg of fresh leaf tissue (Table 2). In general, it was observed that the levels
of protein expression in the ND5873-15 crylac transgenics were higher than levels in
Spunta transgenics. The average protein expression from ND5873-15 transgenics was
0.17 rig/mg of fresh leaf tissue, while it was 0.03 ng/mg of fresh leaf tissue in crylac
Spunta transgenics. The highest expression of Bt—Crylac protein was observed in
ND15.11 and ND15.10. They contained 0.58 ng and 0.32 ng of Bt—Crylac protein/mg of
fresh leaf tissue, respectively. On the other hand, no protein expression was observed
from four Spunta transgenic lines Sp15.04, Sp15.08, Sp15.10 and Sp15.13. The controls,
Spunta and ND5873-15, tested negative for the Bt-Cryl ac protein expression.

Results of Southern blotting showed that the number of inserted cry] ac genes for
both Spunta and ND5873-15 transgenics varied between one to two copies (Figure 3). All
the ND5873-15 crylac transgenics had single insertion except for ND15.10 which had
two copies inserted. Similarly, all the Spunta crylac transgenics had a single gene
insertion except for Spunta15.07 line which has two copies inserted. Southern blot
analysis showed duplicate lines both in ND5873-15 (ND15.05 and ND15.08) and Spunta
transgenic lines (Sp15.01 and Sp15.05). Duplication was also supported by the mortality

data and the protein expression data. The same mortality (55%) was observed in both

21

 

m3 3.3

33 3.3

m3 3b3

m! 3.8

we. 3b—

MESS

3833 02> Evian. €2.00 .3

0—0 Zen—non

203.:
203.5
203.3
203.3
203.3
20333-3

3833 02> 66.36 99.00 3v

Fig. 3. Southern analysis of ND5873-15 and Spunta crylac transgenic lines. DNA was

digested using Xbal and probed with cryIAc gene.

22

Table 2. Mean protein expression of Spunta and ND5873-15 transgenic lines.

 

 

Plant line Ng toxin/mg of fresh
Ieaftissue“

Blank 0.00
ND5873-15 0.00 C
ND15.01 0.05 C
ND15.05 0.06 C
ND15.07 0.10 C
ND15.08 0.04 C
ND15.10 0.32 B
ND15.11 0.58 A
ND15.12 0.01 C
ND15.13 0.01 C
Spunta 0.000 C
Sp15.01 0.02 C
Sp15.02 0.08 C
Sp15.03 0.03 C
SP15.04 0.00 C
Sp15.05 0.06 C
Sp15.06 0.04 C
Sp15.07 0.05 C
Sp15.08 0.00 C
Sp15.10 0.00 C
Sp15.11 0.01 C
Sp15.13 0.00 C

 

* Means with the same letter designation are not signiﬁcantly different as determined by

Fisher’s Protected LSD (a=0.05).

23

ND15.05 and ND15.08. Similarly, the protein expression for both lines was not
signiﬁcantly different from each other. They contained 0.06 and 0.04 ng of Bt-Crylac
protein/mg of fresh leaf tissue, respectively. On the other hand, mortality in Sp15.01
(55%) was not signiﬁcantly different from mortality in Sp15.05 (56%). Also, no
signiﬁcant difference was observed between the protein expression in Sp15.01 (0.02

ng/mg) and Sp15.05 (0.06 ng/mg).

DETACHED LEAF BIOASSAY

The analysis of variance for the percent mortality of potato tuberworm in Spunta
transgenic lines and control showed highly signiﬁcant differences among lines
(P<0.0001). Similarly, signiﬁcant differences among lines (P = 0.02) were observed
between ND5873-15 transgenic lines and control.

Percent potato tuberworm mortality was moderately low (22 %-68 %) for almost
all the cry] ac transgenic lines except for the two lines from ND5873-15 namely ND15.10
and ND15.11 (Figures 4 & 5). These two lines caused 73 % mortality. In general, the
mortality in the Spunta control was signiﬁcantly lower than mortality in the ND5873-15
control and mortality from Spunta transgenics was lower than mortality with ND5873-15
transgenics. The average mortality for Spunta cry] ac transgenic lines was 49% and 57%
for ND5873-15 crylac transgenic lines. Potato tuberworm mortality from all the Spunta
crylac transgenic lines (22% - 58 %) was signiﬁcantly higher than mortality in Spunta (3
%). Potato tuberworm mortality for six ND5873-15 transgenics: ND15.10, ND15.11,
ND15.07, ND15.08, ND15.05 and ND15.01 (36 %-73 %), were signiﬁcantly higher than
mortality with the control ND5873-15 (22 %). Mortality with ND15.12 and ND15.13

lines was not signiﬁcantly different from mortality with the control ND 5873-15.

24

ND15.10 and ND15.11, produced signiﬁcantly higher potato tuberworm mortality
compared with mortality with many of the other ND5873-15 transgenic lines or the
control ND5873-15. There was a signiﬁcant correlation between mortality and protein
expression for the ND lines (r2 = 0.50, P = 0.03) and for the Spunta lines (r2 = 0.40, P =
0.02).

ND15.10 and ND15.11 showed the highest level of control against potato
tuberworm. These two lines were selected and included for further detached-leaf bioassay
evaluation. Unlike the initial detached-leaf bioassay where the evaluation was done after
3 (1, evaluation for the advanced detached-leaf bioassay was after 5 (1. Analysis of
variance for the percent mortality of potato tuberworm in the 5-day detached-leaf
bioassay for ND5873-15 control and ND5873-15 crylac transgenics showed highly
signiﬁcant differences among lines (P<0.0001) (Figure 6). Potato tuberworm mortality in
both ND15. 10 (88%) and ND15.11 (97%) lines was signiﬁcantly higher than mortality in
the control ND5873-15 (17%). Potato tuberworm mortality with ND15.11 was
signiﬁcantly higher than mortality with ND15.10 or the control ND5873-15. Both lines
ND15.10 and ND15.11 expressed the highest levels of Crylac protein and caused the
highest mortality in the detached-leaf bioassay compared with the other Spunta and

ND5873-15 crylac transgenic lines.

TUBER BIOASSAY

Analysis of variance for the percent mortality of potato tuberworm in the tuber
bioassay for ND5873-15 control and ND5873-15 crylac transgenics showed highly
signiﬁcant differences among lines (P<0.0001) (Figure 7). Potato tuberworm mortality in

both ND15.10 (97 %) and ND15.11 (99 %) was signiﬁcantly higher compared with

25

mortality in the ND 5873-15 control (22 %). Potato tuberworm mortality in ND15.10 was
not signiﬁcantly different with mortality in ND 15.11. Signiﬁcantly higher levels of
resistance was observed in both ND15.10 & ND15.11 compared to ND5873-15 in the

tuber & detached-leaf bioassays.

26

70 ——~

 

a a a LSijs = 10.22

 

 

 

(I! ‘3 ’\ N '5 ‘b 0 "b
(0.0 (,9 (,9 (9° (,9 (,9 (,5 {,9 {or ,;> Q
99 9Q 9Q 9Q 9Q 9Q 9Q 9Q 9Q 9Q 9Q 9

Spunta Transgenic lines

Fig 4. Mean percent mortality (detached-leaf bioassay) of ﬁrst instar of potato
tuberworm on Spunta and Spunta transgenic lines. Means with the same letter
designation are not signiﬁcantly different as determined by Fisher’s Protected LSD (at =
0.05).

27

 

   
  

LSDQOS = 17.69

 

 

Sb '0' '3’ '33
0"”. o '
e e

ND5873-15 transgenic lines

Fig. 5. Mean percent mortality (detached-leaf bioassay) of ﬁrst instar of potato
tuberworm on the ND5873-15 and ND5873-15 transgenics. Means with the same letter
designation are not signiﬁcantly different as determined by Fisher’s Protected LSD (or =

0.05).

28

120

 

a LSDQOS = 11.17
100. ..... . ..

Mortality (%) +l-SE

 

 

   

 

 

ND15.11 ND15.10 ND5873-15
N05873-15 transgenic lines

Fig. 6. Mean percent mortality (5-day detached-leaf bioassay) of ﬁrst instar of potato
tuberworm on the control ND5873-15, ND15.10 and ND15.11. Means with the same
letter designation are not signiﬁcantly different as determined by Fisher’s Protected LSD

(on = 0.05).

29

120

 

LSD0_05 = 14.53
100 4

Mortality (%) +/- SE

 

 

 

 

 

 

ND15.11 ND15.10 ND5873-15
N05873-15 transgenic lines

Fig. 7. Mean percent mortality (tuber bioassay) of ﬁrst instar of potato tuberworm on the
ND5873-15 and ND5873-15 transgenic lines. Means with the same letter designation are

not signiﬁcantly different as determined by Fisher’s Protected LSD (or = 0.05) .

30

DISCUSSION

The potato transformation of cry] ac gene using the protocol in our laboratory was
effective in regenerating shoots of transgenic potato lines in both Spunta and ND5873-15
lines. The stringency of selection using the nptII gene was effective for Spunta and
ND5873-15 and made the number of test samples manageable. Although the production
of transgenic shoots from both Spunta and ND5873-15 were successful, NY123 was
found to be resistant to transformation. NY123 was obviously difﬁcult to transform since
the frequency of shoots that rooted in the kanamycin media was very low (13 %).
Furthermore, none from the NY123 shoots tested positive for cry] ac gene via PCR.
Differences in the ease of producing transgenic lines between cultivars were also
observed by Felcher et al. (2003). In their study, they recovered a signiﬁcantly higher
number of transformed Spunta and Atlantic glucose oxidase transgenic lines compared to
Libertas (which gave them few transgenic lines). Similarly, Coombs et al. (2002)
transformed three different potato lines, Yukon Gold, USDA8380-1 and NYL235-4 with
Bt—cry3a, and observed that NYL235-4 produced signiﬁcantly lower number of rooted
shoots in the kanamycin medium compared with the other two lines. NY123 used in our
study, is a progeny of NYL235-4 (W. De Jong, pers. communication). These differences
in the successful regeneration of transgenic shoots between cultivars/lines suggest that
there is genetic variation for regeneration and transformation efﬁciency (Felcher et al.
2003).

Southern blot analysis showed that the number of insertion events in the Spunta
and ND5873-15 crylac transgenic lines were one to two copies. Similar results were

observed by Davidson et al. (2002). They observed that all ﬁve of the highest performing

31

transgenic lines had either one or two copies of crylac9 gene. Douches et al. (1998)
observed copy numbers of Bt-cry5 gene ranging from one to three. Copy numbers
ranging ﬁom one to seven of glucose oxidase gene in transgenic potato lines were
observed from the study conducted by Felcher et al. (2003). Similarly, Beuning et al.
(2001) also observed one to seven copies of cryIac9 gene inserted in tobacco transgenic
lines.

Lower level of protein expression was observed in Sp15.7 as compared with
expression in ND15.10 and ND15.11 Although, Sp15.07 had two inserted crylac genes
it still gave lower level of protein expression compared to ND15.10 (which has also two
inserted crylac genes) and ND15.11 (which has only one inserted crylac gene). In this
study, the number of inserted copies of crylac gene in the plant genome does not
correlate with Crylac protein expression. Similarly, Felcher et al. (2003) observed that
there was no correlation between copy number and gene silencing. Different results were
observed by Davidson et al. (2002), higher protein expression was produced from the
transgenic potato lines with two inserted copies of cry genes as compared with transgenic
potato lines with one inserted gene copy.

The mortality data showed that the highest resistance to potato tuber worm was
observed only from ND15.11 and ND15.10 lines. The high mortality observed from both
lines did correspond to their high Crylac protein expression. The ability to achieve high
protein expression from both lines may be because synthetic codon-modiﬁed cry genes
like crylac, give expression that is signiﬁcantly higher compared to native cry genes
encoding protoxins (Kuvshinov et al. 2001). Native cry genes are expressed poorly in the
transgenic plants, thus the resistance to insect pests is minimal (Beuning et al. 2001).

Protein expressions from cry gene constructs are about 0.1-0.3% of soluble protein

32

corresponding to about 1 pg of toxin protein per lg of fresh leaf tissue (Kuvshinov et al.
2001). In a study conducted by Perlak et al. (1990) as cited by Babu et a1. (2003),
transformed cotton with codon-modiﬁed cryIab and cry] ac gene and observed protein
expression of 0.05-0.1% of the total soluble protein. Variation in the level of resistance to
potato tuberworm was observed from the independently derived Spunta and ND5873-15
crylac transgenic lines. The variation in the level of transgene expression is common
among the population of plants that are independently transformed with the same
transgene. This unpredictable expression is usually attributed to position effects resulting
from the random integration of transgenes into different sites of plant genomes (Davidson
et al. 2002, Conner and Christey 1994).

The levels of protein expressions obtained from this study were higher compared
to the protein expression levels observed by Davidson et al. (2002). They observed that
the amount of Crylac9 protein in all the transgenic potato lines they tested was less than
60 ng/g of fresh leaf tissue. In the study conducted by Kuvshinov et al. (2001) they
observed comparable amount of Cry9Aa protein expression (300 ng/g) in potato plants
and higher Cry9Aa protein expression (1.4 ug/g of leaf material) in tobacco plant.

There was a signiﬁcant correlation between mortality in the feeding assay and the
ELISA data for protein expression for both ND5873-15 and Spunta lines. Variability in
this correlation lies in the variable responses of the insect larvae and also in the
variability inherent in the ELISA determination of protein expression. In a feeding assay,
larvae quit feeding as soon as they became intoxicated. Larvae feeding on potato foliage
with low concentrations of B: may have fed longer and consumed more foliage,
compared to the amount consumed by larvae feeding on foliage with high concentrations

of Bt. Thus, the dose received (amount of consumed foliage X Crylac protein

33

concentration) and the resulting mortality may not have been directly proportional to Bt
concentration. Mortality in the transgenic lines is not only dependent on the amount of
protein expressed in the leaves but is also inﬂuenced by the total amount of Cry] ac
protein ingested by the potato tuberworm.

The resistance provided by the integration of cry] ac gene in this study, up to 97
% potato tuberworm mortality, was similar to the resistance provided by the integration
of Bt-cryS gene in the study conducted by Westedt et al. (1998). They observed that
potato transformation with a codon-modiﬁed Bt-cry5 gene provided resistance against
potato tuberworm of up to 96% mortality. In the study conducted by Mohammed et al.
(2000), they observed higher potato tuberworm mortality of up to 100 % in the same Bt-
cry5 Spunta transgenic lines. Similarly, 100 % potato tuberworm mortality after 10 d was
observed with both crylac and cry] ab potato transgenic lines (Davidson et al. 2002).
Jansens et al. (1995) observed protein content of 3-118 ng toxin/mg total protein in the
leaves in the Yesmina and Kennebee cry] ab transgenic lines causing 40 %-100 % potato
tuberworm mortality.

Higher mortality observed in the ND5873-15 control compared with mortality in
the Spunta control is probably the result of natural insect resistance factors
(glycoalkaloids) incorporated into the ND5873-15 (Lachman et al. 2001). The low
mortality in the ND5873-15 (22 %) in spite of the fact that it has natural resistance
(glycoalkaloids) may be attributed to the level of glycoalkaloids content in the leaves
used in the detached-leaf bioassay. The glycoalkaloids concentration in potato plants can
be highly affected by the temperature, light intensity and day length. Glycoalkaloids are
present at higher concentration in the aerial (leaves, stems and sprouts) part of the potato

plant and are normally present in lower concentration in the tubers (Lachman et al. 2001).

34

Similarly, Laﬁa and Lorenzen (2000) observed a signiﬁcant increase in foliar
glycoalkaloids when plants were grown at higher temperature (32/27 °C).

Higher mortality observed in the 5-day detached-leaf bioassay with ND15.10 &
ND15.11 compared with the mortality on the initial detached-leaf bioassay can be
attributed to the additional 2 d. Longer exposure of the potato tuberworm larvae to the
protein/toxins could lead to higher toxin ingestion and higher mortality. Running the
detached-leaf bioassay for 7-10 d is highly recommended to ensure longer exposure of
potato tuberworm to the toxin. Results from the study conducted by Davidson et al.
(2002) suggests that running the detached-leaf bioassay for up to 10 days could give
higher potato tuberworm mortality of up to 100%. For the Cry] ac protein to have
maximum effect in the Spunta and ND5873-15 transgenic lines, it may require exposure
time of up to 7-10 d.

The production of more independently selected transgenic lines will allow
recovery of several lines with high transgene expression, as well as phenotypically
normal apprearance and yield performance (Meiyalaghan et al. 2004, Conner and
Christey 1994). Field and storage trials are also important and necessary tests in the
identiﬁcation of high performing transgenic potato lines with good transgene expression,
phenotypic appearance and yield equal to that of the commercial cultivars in the market.

Further study on the inheritance of my] ac gene in their progeny is also interesting
since (Babu et al. 2003) it is common that the genes showing expression in transgenic
plants are stably inherited into the progeny without detrimental effects on the recipient
plant.

The results of this study showed that combining natural host plant resistance and

engineered resistance with the Bacillus thuringiensis-crylac gene in potato plant was

35

effective in conferring increase resistance to potato tuberworm in detached-leaf
bioassays. The cryI ac gene can be another source of resistance which can be pyramided

with other genes for host plant resistance to potato tuberworm in potato.

36

APPENDIX A

Table 3. DNA concentrations used in the PCR reaction.

 

 

Lines Concentration (ng/ul)
Spunta l8
Sp15.01 11
Sp15.02 18
Sp15.03 30
Sp15.04 30
Sp15.05 31
Sp15.06 76
Sp15.07 47
Sp15.08 49
Sp15.10 23
Sp15.11 104
Sp15.13 59
ND5873-15 4
ND15.01 23
ND15.05 15
ND15.07 l6
ND15.08 33
ND15.10 16
ND15.11 12
ND15.12 14
ND15.13 18

 

37

APPENDIX B

Table 4. Total glycoalkaloids contents of ND5873-15, ND5873-15 transgenic lines,

Spunta and Spunta transgenic lines.

 

 

 

 

TGA Leptinines Leptines Solanine Chaconine Unknown
Clones mglg °/o °/o % % °/o
Spunta 5.33 0.00 0.00 7.25 29.19 63.56
SP15.01 2.49 0.00 0.00 18.58 81.42 0.00
SP15.02 11.61 0.00 0.00 3.99 22.90 73.11
SP15.03 7.58 0.00 0.00 7.94 36.42 55.64
SP15.04 8.04 0.00 0.00 7.82 40.36 51.82
SP15.05 8.39 0.00 0.00 4.14 22.17 73.68
SP15.06 7.74 0.00 0.00 7.06 37.49 55.45
SP15.07 7.06 0.00 0.00 8.99 42.77 48.24
SP15.08 13.14 0.00 0.00 3.99 19.19 76.82
SP15.10 5.79 0.00 0.00 8.10 40.61 51.29
SP15.11 4.82 0.00 0.00 10.42 38.26 51.32
SP15.13 4.18 0.00 0.00 6.34 22.32 71.33
ND5873.15 8.88 26.21 19.38 21.37 33.04 0.00
ND15.01 24.25 26.87 33.58 17.43 22.12 0.00
ND15.05 14.66 10.80 34.61 21.82 32.77 0.00
ND15.07 29.89 8.32 40.83 20.99 29.86 0.00
ND15.08 27.24 9.42 28.39 23.61 38.58 0.00
ND15.10 18.21 14.36 34.11 17.99 33.54 0.00
ND15.11 13.18 27.30 41.88 11.98 18.84 0.00
ND15.12 28.63 12.87 44.30 17.09 25.74 0.00
ND15.13 22.56 19.59 36.10 16.65 27.66 0.00

 

 

 

 

 

 

 

38

 

LITERATURE CITED

Babu, R., A. Sajeena, K. Seetharaman and M. Reddy (2003). Advances in genetically
engineered (transgenic) plants in pest management-an over view. Crop Protection
22:1071-1086.

Barton, K. and M. Miller (1993) Production of Bacillus thuringiensis insecticidal proteins
in plants. Transgenic plants 1: 297-315.

Belknap, W., D. Corsini, J. Pavek, G. W. Snyder and D. R. Rockhold (1994). Field
performance of transgenic potatoes. The molecular and Cellular Biology of the Potato,
2nd ed. pp. 233-243.

Beuning, L., D.S. Mitra, N.P. Markwick and AP Gleave (2001). Minor modiﬁcations to
the crylAc9 nucleotide sequence are suﬁ'rcient to generate transgenic plants resistant to
Phthorimaea operculella. Ann. Appl. Biol. 138:281-292.

Capinera, J. L. (2001) Handbook of vegetable pests. Academic Press, San Diego Ca.,
pp.359-362.

Chan, M., L. Chen, and H. Chang (1996). Expression of Bacillus thuringiensis (B.t.)
insecticidal crystal protein gene in transgenic potato. Bot. Bull. Acad. Sin. 37: 17-23.

Conner, A. J., M. K. Williams, D.J. Abernethy, P. J. Fletcher and R. A. Genet (1994).
Field performance of transgenic potatoes. New Zealand Journal of Crop & Horticultural
Science 22:361-371.

Coombs, J ., D.S. Douches, W. Li, B. Graﬁus and W. Pett (2002). Combining engineered
(Bt-cry3A) and natural resistance mechanisms in potato for control of Colorado potato
beetle. J. Amer. Soc. Hort. Sci. 127(1):62-68.

Davidson, M., J. Jacobs, J. Reader and R. Butler (2002). Development and evaluation of
potatoes transgenic for a crylAc9 gene conferring resistance to potato tubennoth. J.
Amer. Soc. Hort. Sci. 127(4):590-596.

Dempsey, D. A., H. Silva and D. Klessig (1998). Engineering disease and pest resistance
in plants. Trends in Microbiology 56(2):54-6l.

Douches, D.S., D. Maas, K. Jastrzebski and R.W. Chase (1996). Assesment of potato
breeding progress in the USA over the last century. Crop Sci. 36: 1544-1552.

Douches, D. S., A.L. Westedt, K. Zarka and B. Schroeter (1998). Potato transformation

to combine natural and engineered resistance for controlling tuber moth. HortScience
33(6): 1053-1056.

39

Douches, D., T. J. Kisha, J. J. Coombs, W. Li, W. L. Pett and E]. Graﬁus (2001).
Effectiveness of natural and engineered host plant resistance in potato to the Colorado
potato beetle. HortScience 36(5):967-970.

Felcher, K., D. Douches, W. Kirk, R. Hammerschmidt and W. Li (2003). Expression of a
fungal glucose oxidase gene in three potato cultivars with different susceptibility to late
blight (Phytophthora infestans Mont. deBary). J. Amer. Soc. Hort. Sci. 128(2):238-245.

Ferry, N., M. Edwards, J. Gatehouse and A. Gatehouse (2004). Plant-insect interactions:
molecular approaches to insect resistance. Current Opinion in Biotech. 15:155-161.

Flanders, K., S. Amone and E. Radcliffe (1999). The potato: Genetic resources and insect
resistance. Global plant genetic resource for insect-resistant crops. pp. 207-239.

Gould, F., P. Follet, B. Nault and G. G. Kennedy ( 1994). Resistance management
strategies for transgenic potato plants, p. 255-277. In: G.W. Zehnder, M.L. Powelson,

R.K. Jansson, and K.V. Raman (eds.). Advances in potato pest biology and management.
APS Press, St. Paul. Minn.

Gebhardt, C. and J. Valkonen (2001). Organization of genes controlling disease
resistance in the potato genome. Annu. Rev. Phytopathol. 39:79-102.

Gill, S., E.A. Cowles and RV. Pietrantonio (1992). The mode of action of Bacillus
thuringiensis endotoxins. Annu. Rev. Entomol. 37:615-636.

Head, G., J. Surber, J. Watson and J. Martin (2002). No detection of CryIAc protein in
soil after multiple years of transgenic Bt cotton (Bollgard) use. Environ. Entomol. 31(1):
30-36.

Jansens, S., M. Comellissen, R. De Clerco, A. Reynaerts and M. Peferoen (1995).
Phthorr'maea operculella (LepidopterazGelechiidae) resistance in potato by expression of

the Bacillus thuringiensis CryIab insecticidal crystal protein. Ent. Soc. of Amer.
88(5): 1469-1476.

Jerkins, J. and D. H. Dean (2001). The complex receptor-binding interactions of
insecticidal proteins from Bacillus thuringiensis. BIAJournal 1: 25-27.

Kalazich, J. C., and R. L. Plaisted (1991).Association between trichome characters and

agronomic traits in Solanum tuberosum (L. ) x S. berthaultr’i (Hawkes) hybrids.American
Pot. Jour. 68:833-847.

Kuvshinov, V., K. Koiva, A. Kanerva and E. Pehu (2001). Transgenic crop plants
expressing synthethic cry9Aa gene are protected against insect damage. Plant Sci.
160:341-353.

40

Lachman, J ., K. Hamouz, M. Orsék and V. Pivec (2001). Potato glycoalkaloids and their
signiﬁcance in plant protection and human nutrition - Review. Series Rostlinna Vyroba
47(4):181-191.

Laﬁa, A. and J. H. Lorenzen (2000). Inﬂuence of high temperature and reduced
irradiance on glycoalkaloid levels in potato leaves. J. Amer. Soc. Hort. Sci. 125(5):563-
566.

Lagnaoui, A., V. Caﬁedo and D.S. Douches (2000). Evaluation of Bt-crylla] (cryV)
transgenic potatoes on two species of potato tuber moth, Phthorimaea operculella and

Symmetrischema tangolias (Lepidoptera: Gelechiidae) in Peru. CIP Program Report
1999-2000. 117-121.

Lapointe, S. and W. Tingey (1986). Glandular trichomes of Solanum neocardenasii
confer resistance to green peach aphid (HomopterazAphididae). Jour. of econ. Entom.
70(5): 1264- l 268.

Lawson, D. R., R. Veilleux and A. R. Miller (1993). Biochemistry and genetics of
Solanum chacoense steroidal alkaloids: Natural resistance factors to the Colorado potato
beetle. Current topics in Bot. Research 12335-351.

Meiyalaghan, 8., MM. Davidson, M.G.F. Takla, S.D. Wratten and AJ. Conner (2004).
Effectiveness of four cry genes in transgenic potato conferring resistance to potato
tuberrnoth. New directions for a diverse planet: Proceedings of the 4th International Crop
Science Congress. 26Sept — lOct 2004.

Mohammed, A., D.S. Douches, W. Pett, E. Graﬁus, J. Coombs, Liswidowati, W.Li and
MA. Madkour (2000) Evaluation of potato tuber moth (Lepidoptera: Gelechiidae)
resistance in tubers of Bt-cry5 transgenic potato lines. J .Econ. Entom. 93(2): 472-476.

Naimov, S., S. Dukiandjiev and R. de Maagd (2003). A hybrid Bacillus thuringiensis
delta-endotoxin gives resistance against a coleopteran and a lepidopteran pest in
transgenic potato. Plant Biotech. Jour. 1:51-57.

SAS (2001). The SAS System for Windows. Software release 6.12. SAS Institute, Inc.
Cary, NC, USA.

Stoner, Kimberly (1996). Plant resistance to insects: A resource available for sustainable
agriculture. Biol. Agric. & Hort. 1327-38.

Tingey, Ward M. (1984). Glycoalkaloids as pest resistance factors. American Potato
Journal 61:157-167.

Theunis, W., R. M. Aguda, W. T. Cruz, C. Decock, M. Peferoen, B. Lambert, D.G.
Bottrell, F.L. Gould, J.A. Litsinger and ME. Cohen (1998). Bacillus thuringiensis
isolates from the Philippines: habitat distribution, delta-endotoxin diversity, and toxicity
to rice stern borers (Lepidoptera: Pyralydae). Bull. Entom. Res. 88:335-342.

41

Westedt, A. L., D.S. Douches, W. Pett and E]. Graﬁus (1998). Evaluation of natural and
Engineered resistance mechanisms in Solanum tuberosum for resistance to Phthorimaea
operculella (LepidopterazGelechiidae). J .Econ. Entomol. 91(2): 552-556.

Yadav, NR. and M. Sticklen (1995). Direct and efﬁcient plant regeneration from leaf
explants of Solanum tuberosum 1. cv. Bintje. Plant Cell Reports. 14:645-647.

Yencho, G. C. and W. Tingey (1994). Glandular trichomes of Solanum berthaultr'i alter

host preference of the Colorado potato beetle, Leptinotarsa decemlineata. Entomol. Exp.
Appl. 70:217-225.

42