Journal of Experimental Botany, Vol. 54, No. 383, pp.

835±844, February 2003

DOI: 10.1093/jxb/erg071

RESEARCH PAPER

Transgenic loblolly pine (Pinus taeda L.) plants

expressing a modi®ed d-endotoxin gene of Bacillus
thuringiensis with enhanced resistance to Dendrolimus
punctatus Walker and Crypyothelea formosicola Staud

Wei Tang1,3 and Yingchuan Tian2

1
North Carolina State University, Forest Biotechnology Group, Centennial Campus, PO Box 7247, Raleigh,
NC 27695-7247, USA

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2
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China

Received 4 April 2002; Accepted 30 September 2002

Abstract transgenic plants recovered could represent a good

A synthetic version of the CRY1Ac gene of Bacillus
thuringiensis has been used for the transformation of
loblolly pine (Pinus taeda L.) using particle bombard-
ment. Mature zygotic embryos were used to be bom-
barded and to generate organogenic callus and
transgenic regenerated plants. Expression vector
pB48.215 DNA contained a synthetic Bacillus thurin-
giensis (B.t.) CRY1Ac coding sequence ¯anked by
the double cauli¯ower mosaic virus (CaMV) 35S pro-
moter and nopaline synthase (NOS) terminator
sequences, and the neomycin phosphotransferase II
(NPTII) gene controlled by the promoter of the nopa-
line synthase gene was introduced into loblolly pine
tissues by particle bombardment. The transformed
tissues were proliferated and selected on media with
kanamycin. Shoot regeneration was induced from the
kanamycin-resistant calli, and transgenic plantlets
were then produced. More than 60 transformed plants
from independent transformation events were
obtained for each loblolly pine genotype tested. The
integration and expression of the introduced genes
in the transgenic loblolly pine plants was con®rmed
by polymerase chain reactions (PCR) analysis, by
Southern hybridization, by Northern blot analysis,
and by Western blot analysis. Effective resistance of
transgenic plants against Dendrolimus punctatus
Walker and Crypyothelea formosicola Staud was veri-
®ed in feeding bioassays with the insects. The
3
To whom correspondence should be addressed. Fax: +1 919 515 7801. E-mail: wei_tang@ncsu.edu
Abbreviations: BA, benzyladenine; B.t., Bacillus thuringiensis; CaMV, cauli¯ower mosaic virus; 2,4-D, 2,4-dichlorophenoxyacetic acid; IBA, indole-3-butyric
acid; NOS, nopaline synthase; NPTII, neomycin phosphotransferase II gene; PCR, polymerase chain reactions.
opportunity to analyse the impact of genetic engin-
eering of pine for sustainable resistance to pests
using a B. thuringiensis insecticidal protein. This
protocol enabled the routine transformation of
loblolly pine plants that were previously dif®cult to
transform.
Key words: Bacillus thuringiensis (B.t.) CRY1Ac, biolistic
transformation, insect feeding bioassay, Pinus taeda L.
Introduction
Loblolly pine (Pinus taeda L.) is one of the most widely
planted forest species in both tropical and subtropical
regions and an important tree in commercial forestry
worldwide. Dendrolimus punctatus Walker and
Crypyothelea formosicola Staud are two of the major
pests threatening the wood production of loblolly pine.
Systemic chemical pesticides for these insect pests are
usually harmful, and the implementation of an environ-
mentally friendly way of controlling this pest would be a
preferable option (Huang et al., 2002). Compared to
chemicals, insecticidal proteins do not work as contact or
external poisons, but instead as internal poisons.
Therefore, the transgenic approach would be a valuable
strategy for the control of these insect pests because the
insecticidal protein was eaten by the pests (Sardana et al.,
1996; Cho et al., 2001). As the use of Bacillus

Journal of Experimental Botany, Vol. 54, No. 383, ã Society for Experimental Biology 2003; all rights reserved
836 Tang and Tian
thuringiensis genes to transform plants for protection
against insect pests is currently considered to be the most
reliable strategy (Estruch et al., 1997; Schuler et al., 1998),
investigations were initiated to determine the susceptibility
of insect pests to B. thuringiensis insecticidal proteins and
to identify the candidate genes for the transformation of
plants (Guerreiro et al., 1998). To date, six different
endotoxins of B. thuringiensis have been tested against
pests (Guerreiro et al., 1998), and the toxin expressed by
the cry1Ac gene, widely used to confer resistance to
lepidopterae (Dandekar et al., 1998), has been demon-
strated to be the most effective.
Conventional breeding of woody species is a low-
ef®ciency and time-consuming process due to their long
life cycles. Genetic engineering could alleviate these
problems by incorporating known genes into elite genetic
backgrounds. Genetic modi®cation of pine for insect,
disease, and stress resistance, lignin content, and the
improvement of wood quality is thus of major commercial
importance. Pine improvement by conventional plant
breeding methods has been limited, mainly due to the
prohibitively long reproduction cycles and slow seed
maturation of these plants. Therefore, the application of
genetic engineering techniques to pine improvement
appears to be an attractive alternative. Since transgenic
plants from microprojectile-mediated gene transfer have
been reported for rice (Alam et al., 1998; Christou et al.,
1991; Datta et al., 1998), soybean (Christou et al., 1989),
papaya (Fitch et al., 1990), potato (Perlak et al., 1993),
tobacco (Iida et al., 1991), and poplar (Han et al., 1997;
McCown et al., 1991), potential progress has been made in
plant genetic transformation (Franche et al., 1997; Schuler
et al., 1998). At present, the regeneration of transgenic
conifers via Agrobacterium-mediated transformation has
been reported for Larix decidua (Huang et al., 1991), larch
(Shin et al., 1994), hybrid larch (Larix kaempferi3L.
decidua) (Levee et al., 1997), white pine (Pinus strobus L.)
(Levee et al., 1999), Norway spruce (Picea abies L.)
(Wenck et al., 1999), white spruce (Picea glauca) (Le
et al., 2001), and loblolly pine (Tang et al., 2001). Stable
transformation and regeneration of transgenic conifers
using biolistics has been achieved for Picea abies (Walter
et al., 1999), Larix laricina (Klimaszewska et al., 1997),
Picea glauca (Ellis et al., 1993), Picea mairana (Charest
et al., 1996), and Pinus radiata (Walter et al., 1998).
Although transformation protocols are available for pine
(Birch, 1997; Humara et al., 1999; Tz®ra et al., 1996),
there is no report available on the stable genetic trans-
formation of loblolly pine by particle bombardment.
In this article, the ®rst transformation of loblolly pine
using particle bombardment for expression of an agro-
nomic trait is reported. In the investigation reported here, a
synthetic CRY1Ac gene was introduced into three loblolly
pine genotypes. The data revealed the correct integration
and expression of the CRY1Ac gene within the loblolly
pine genome. Furthermore, there was a good correlation
between CRY1Ac gene expression and insect bioassays
performed on transformed plants. In this investigation, a
reproducible method for the gene transfer and regeneration
of transformed plants from the loblolly pine mature
zygotic embryos using particle bombardment was estab-
lished. A key to develop this optional genetic transform-
ation protocol is to use mature zygotic embryos of different
genotypes of loblolly pine as targeting tissues. In this
study, transgenic loblolly pine plants with enhanced
resistance to Dendrolimus punctatus Walker and
Crypyothelea formosicola Staud were evaluated.
Materials and methods
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Plant materials and plasmid construction
Mature seeds of three loblolly pine genotypes were used for
transformation experiments, one was collected from Zhangjiajie
Loblolly Pine Seed Orchard (genotype J-29, provided by Professor
Dongxiang Xu, Hunan Province, China), and two were from Yingde
Loblolly Pine Orchard (genotypes E-11 and E-44, provided by
Professor Weihua Zhong) in October 1996. Seeds were stored in
plastic bags at 4 °C before they were used for tissue culture. Seeds
were disinfected by immersion in 70% ethyl alcohol for 30 s and in
0.1% mercuric chloride for 10±15 min, followed by ®ve rinses in
sterile distilled water. Mature zygotic embryos were aseptically
removed from the megagametophytes and placed horizontally on a
solidi®ed callus induction medium in 125 ml Erlenmeyer ¯asks or
153100 mm Petri dishes. Mature zygotic embryo explants were
used for transformation experiments after being cultured on a
pretreatment medium that consisted of TE medium (Tang et al.,
2001) supplemented with 10 mg l±1 2,4-dichlorophenoxyacetic acid
(2,4-D), 4 mg l±1 benzyladenine (BA), and 4 mg l±1 kinetin for 3 d.
T-DNA of plasmid pB48.215 (Fig. 1) contained two genes, one is
a synthetic Bacillus thuringiensis (B.t.) CRY1Ac coding sequence
¯anked by the double cauli¯ower mosaic virus (CaMV) 35S
promoter (Kay et al., 1987) and nopaline synthase (NOS) terminator
sequences, and the other is a selectable marker gene, neomycin
phosphotransferase II (NPTII) controlled by the promoter of the
nopaline synthase gene (Li et al., 1994). Plasmid construction was
produced following the procedure reported by Li et al. (1994). The
double CaMV 35S promoter and translation enhancer fragments
(Gallie and Kado, 1989) cut by HindIII and BamHI from pD511
were cloned to pBin437 to produce pBin438 (Li et al., 1994). The
Bacillus thuringiensis (B.t.) CRY1Ac gene fragments cut by BamHI
and SaII from pB48.103 (Li et al., 1994) were inserted in pBin438 to
produce pB48.215 (Li et al., 1994). Plasmid pB48.215 were
transferred into E. coli strain DH5a and bacteria were cultured on
LB medium (Sambrook et al., 1989) with 100 mg l±1 carbenicillin
and 50 mg l±1 kanamycin. Plasmid pB48.215 were largely isolated
from bacterium cultures grown overnight at 37 °C in liquid LB
medium (Sambrook et al., 1989) supplemented with 100 mg l±1
carbenicillin and 50 mg l±1 kanamycin and puri®ed by CsCl ethidium
bromide density gradient centrifugation (Sambrook et al., 1989), and
then used for biolistic transformation.
Transformation and regeneration of transgenic plants
Microprojectile bombardment was carried out using the Biolistic
PDS 1000/He System (Bio-Rad, Hercules, Calif.) at 1100 psi
following the manufacturer's instructions and protocol. All access-
ible parts of this equipment were surface-sterilized using 70% ethyl
alcohol 15 min before use. Microcarriers were prepared as follows: 3
 Genetic transformation of loblolly pine 837

Fig. 1. Linear plasmid map indicating the localization of the different genes (nptII neomycin phosphotransferase gene, B.t. a synthetic Bacillus
thuringiensis CRY1Ac coding sequence), promoters (nosPro the promoter of nopaline synthase gene, 2335SPro the double cauli¯ower mosaic
virus 35S promoter), terminators (nosTer the terminator from nopaline synthase gene), W translation enhancer, and T-DNA borders (LB left
border, and RB right border). The arrows indicate the translation orientation of the genes. The probe used in Southern blot analysis of transgenic
plants is the restriction enzymes BamHI and SalI fragment of B.t. gene, HindIII was used to digest genomic DNA isolated from transgenic plants,
and their positions are indicated immediately above. Binding sites of PCR primers nrp and nfp are shown as black rectangles.

mg gold (0.6, 1.0, and 1.1 mm) particles, suspended in 50 ml distilled Genomic DNA Isolation Kit (Sigma) following the manufacturer's
water, were mixed with 20 mg DNA of plasmid, followed by the protocol. These transgenic plants were 8±10 cm in height and

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addition of 175 ml CaCl2 (2.5M) and 70 ml spermidine (0.1 M,
Sigma). The mixture was gently vortexed for 10 mix at room
temperature and then centrifuged brie¯y. The gold particles were
washed twice with 100% ethanol and resuspended in 100% ethanol
(50 ml). Fifteen microlitres microcarrier suspension was used for
each bombardment. Explants were placed on the TE callus induction
medium (Tang et al., 2001) for 3 d before bombardment.
Bombardment was performed with a rupture disc pressure of 1100,
1350, and 1550 psi, respectively, a gap distance of 1.5 cm, a
macrocarrier travel distance of 8 mm, and target distance of 12 cm.
After bombardment, embryos of all three genotypes were transferred
to TE callus induction medium (Tang et al., 2001). After a period of
21±28 d, they were transferred to a selective TE medium supple-
mented with 15 mg l±1 of kanamycin. Embryos were transferred to
new selective medium every 3 weeks. After calli were formed from
embryos, calli were subcultured every 14 d. Kanamycin-resistant
calli were maintained on selection medium for longer than was
practically required for regeneration, to ensure that a true frequency
for kanamycin resistance was established. Approximately 1 mg DNA
was introduced per bombardment. Similar samples of all three
families that were not bombarded and bombarded with gold particles
without DNA served as the controls. Treatments were replicated
three times.
Organogenic calli were initiated from transformed mature zygotic
embryos cultured on TE induction medium (Tang et al., 2001) with
15 mg l±1 kanamycin. Plant regeneration was carried out on
differentiation medium supplemented with selection pressure
according to a procedure previously described (Tang and Ouyang,
1999), i.e. precise composition of the medium. All media were
supplemented with 3% sucrose and 0.3% Phytagel (Sigma). Media
were adjusted to pH 5.8 prior to autoclaving 20 min at 121 °C. All
cultures were cultured at 25 °C in a culture room. Adventitious shoot
induction was conducted in the dark and adventitious shoot
differentiation, proliferation and rooting were conducted at 25 °C
under a 16 h photoperiod with cool ¯uorescent light (100 mmol m±2
s±1). Differentiation was evaluated by the percentage of calli forming
transgenic adventitious shoots. The acclimatization of regenerated
plantlets was conducted by decreasing the relative humidity to
ambient conditions over a period of 1 week, and then the plantlets
were established in soil. The frequency of calli forming transgenic
shoots and the frequency of transgenic shoots forming roots were
determined in the 9th week of culture. The data were analysed by the
Analysis of Variance (ANOVA).
Polymerase chain reaction (PCR) analysis
Loblolly pine genomic DNA was extracted from 300±500 mg fresh
needle tissue of control and putative transgenic plants using a
established in soil for one year in a greenhouse, respectively. The
primer set utilized for ampli®cation of insert DNA was the nptII
forward primer (nfp) 5¢-ACAAACAGACAATCGGCTGC-3¢ and
reverse primer (nrp) 5¢-AAGAACTCGTCAAGAAGGCG-3¢. A
total of 100±300 ng of genomic DNA was used as the template in
a 50 ml PCR reaction mix containing 200 mM each of dATP, dCTP,
dGTP, and dTTP, 35 pmol of each primer, 2.5 U Taq DNA
polymerase (Promega), 1.5 mM MgCl2, and 5 ml 103 buffer [500
mM KCl, 100 mM Tris-HCl (pH 9.0 at 25 °C), 1% Triton X-100, 15
mM MgCl2]. The reaction proceeded in a programmable MJ
MiniCycler (MJ Research, Watertown, Mass., USA). The PCR
conditions were 95 °C for 5 min followed by 30 cycles of 95 °C for
30 s, 52 °C for 1 min, and 72 °C for 2 min. Cycling was followed
with a ®nal incubation of 72 °C for 10 min. PCR products were
observed under UV after electrophoresis on a 1.0% agarose gel with
0.1% ethidium bromide. Molecular marker is 1-kb DNA marker
(Gibco-BRL).
Southern hybridization
Total genomic DNA was isolated from transgenic plants by using a
Genomic DNA Isolation Kit (Sigma) following the manufacturer's
protocol. Twenty mg of total genomic DNA from putative transgenic
and non-transformed control plants was digested overnight with the
restriction enzyme HindIII (Boehringer Mannheim) at 37 °C.
Digested DNA of each line were separated through a 1% agarose
gel prepared in 13 TBE (Sambrook et al., 1989) and fragments were
transferred from the agarose gels to a nylon membrane (Hybond-N;
Amersham) and cross-linked to the membrane by UV using
Stratalinker (1200 Joules 100; Stratagene). The DNA ®xed on
membranes was hybridized (at 65 °C) with the Bacillus thuringiensis
(B.t.) CRY1Ac gene probe (BamHI and SacI (Boehringer
Mannheim) fragment of B.t. gene), which was labelled with
b-[32P]dCTP (Ready to Go Labeling Beads (Pharmacia)).
Hybridization was performed according to Sambrook et al. (1989)
in 53 SSPE, 53 Denhardt's solution, 0.1% SDS, 0.1 mg ml±1
denatured ®sh sperm, 0.1 g ml±1 dextran sulphate for 20 h before the
membranes were washed with 0.13 SSPE, 0.1% SDS solution at 65
°C. Hybridizing bands were detected by exposure to Kodak
X-OMAT autoradiography ®lms at ±80 °C for 3 d.
Isolation of RNA and Northern blot analysis
Ribonucleic acid was extracted from frozen leaf tissue as previously
described (Chang et al., 1993). Needle samples were collected from
PCR and Southern-positive transgenic loblolly pine plants and
immediately frozen in liquid nitrogen and stored at ±80 °C. Samples
of RNA were extracted from 1.5 g of needle tissue using the
procedure of Chang et al. (1993). The isolated RNA was precipitated
838 Tang and Tian
twice with 4 M LiCl for 60 min at 0 °C to remove traces of DNA and
small RNA species. Denatured total RNA samples were fractionated
by 1.2% formaldehyde agarose gel electrophoresis (1.5% [w/v]
agarose) and then capillary blotted onto `Zeta Probe' blotting
membranes (Bio-Rad). The blot was probed using a B.t. probe
fragment labeled with b-[32P]dCTP. Conditions for hybridization
and washing of blots were those recommended for use (Sambrook
et al., 1989). Hybridization of the probe DNAs to the blot was
recorded on blue-sensitive X-rayâ ®lm. The integrity and the amount
of RNA applied to each lane were veri®ed by ethidium bromide
staining.
Western blot analysis
Plant tissues (1 g fresh weight) were ground with a microfuge pestle
in 2 vols of ice-cold 50 mM Hepes buffer (pH 7.0). The slurry was
centrifuged for 10 min (13 000 g) in a microcentrifuge at 4 °C and
the supernatant was collected. The protein concentration was
determined using a protein dye-binding assay kit (Bio-Rad). The
supernatant solutions were heat-denatured at 100 °C for 5 min after
addition of an equal volume of 23 SDS sample buffer [100 mM
Tris-Cl (pH 6.8), 200 mM dithiothreitol, 4% SDS, 0.01%
bromophenol blue, and 20% glycerol]. Analysis by SDS-PAGE
was carried out on 10% SDS-polyacrylamide gels according to
Sambrook et al. (1989). Prestained molecular-weight standards
(broad range; Bio-Rad) were used as markers. The separate proteins
were transferred to nitrocelluloseâ®lters (Bio-Rad) by semi-dry
electroblotting for 25 min at 25 V with transfer buffer [48 mM TRIS-
Cl (pH 9.2), 39 mM glycine, 10% SDS, and 20% methanol].
Immunodetection was carried out using anti-B.t. antiserum
(Clontech) at a 1:2000 dilution in TBS [100 mM Tris-Cl (pH 7.4)
and 0.9% NaCl]. Alkaline phosphatase-conjugated anti-rabbit IgG
(Sigma Chemicals) was used as the secondary antibody at 1:5000
dilution in TBS and this was detected using nitroblue tetrazolium
and 5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad).
Insect bioassays
The eggs of Dendrolimus punctatus Walker and Crypyothelea
formosicola Staud were collected in 1998 from loblolly pine plants
at the Zhangjiajie Loblolly Pine Orchard, Hunan province, China
and then were kept in an environmental chamber at 2% relative
humidity, and under a photoperiod of 16:8 h (light:dark) (Shelton
et al., 1991; Metz et al., 1995; Cao et al., 1999). For tests of
transgenic calli, a needle containing 15±20 eggs was placed on
transgenic calli maintained in containers of 1% water agar under a
16:8 h (light:dark) regime at 25 °C. In some cases, transgenic calli
were inoculated with freshly hatched larvae instead of eggs. Larval
mortality was scored after 7 d. For assays of transgenic plants, all
positive transgenic plants expressing the CRY1Ac protein were
tested indiviually for lethality and growth retardation of
Dendrolimus punctatus Walker and Crypyothelea formosicola
Staud larvaes using a whole plant-feeding assay. The test plants
were individually infested with larvaes of 3±5 mm in length for 7 d at
25 °C under a 16/8 h light/dark regime. Transgenic plants were
placed on a piece of moistened ®lter paper in a Petri dish (1003200
mm). Larvae were placed on the shoot, and the Petri dish was sealed
with masking tape. After 7 d, the number of alive and dead larvae
was determined for each Petri dish. A similar infestation was also
done for control plants. The control plants are untransformed
regenerants of the same age as the transformed ones. The bioassay
was replicated three times for each plant. The mortality rate was
expressed as a percentage (the number of dead larvae/number of
larvae applied) 3 100%. The data were analysed by the Analysis of
Variance (ANOVA).
Results
Transformation, selection, and regeneration of
transgenic plants
Bombarded embryos were ®rst induced to form calli by
being incubated in a medium without kanamycin for 28 d.
The use of the preculture on TE callus induction medium
(Tang et al., 2001) for 28 d produced a signi®cantly higher
number of callus than did the control. The initial 28 d of
culture without kanamycin selection was intended to
promote active proliferation of calli on the surface of the
bombarded embryos. The optimal concentration for select-
ing transformed cells and tissues was determined by
culturing mature zygotic embryos on callus induction
medium containing different concentration of kanamycin.
According to the preliminary experiments, 15 mg l±1
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kanamycin was used to identify transformed cells and
tissues. Two to three weeks after bombarded mature
zygotic embryos were transferred onto callus induction
medium supplemented with 15 mg l±1 kanamycin,
kanamycin-resistant calli were observed. Callus was
formed on cotyledons, hypocotyls, and radicles of
embryos. Proliferation of transgenic calli was achieved
by subcultured kanamycin-resistant calli on fresh callus
induction medium with kanamycin. This sequential
manipulation was critical for transformation success as
prior attempts to obtain transformants from embryos
transferred directly to TE medium containing kanamycin
after bombardment failed.
Experiments were completed involving between 1000
and 9000 zygotic embryos for each genotype. On a
selective medium with 15 mg l±1 of kanamycin, calli
appeared on the surface of the necrotic embryos. After
about two months of culture, such calli occurred on
approximately 5% of the zygotic embryos. For the
genotype J-29, small groups of calli appeared on 2±5%
of the zygotic embryos, with considerable variability. On
the genotype E-11, less than 1% of the explants produced
calli. While the transformation process has been improved
with further experimentation, it remained dif®cult to
obtain transformed calli for each experiment; most of the
results were obtained from 5±10 transformation assays for
the E-11 and E-44 genotypes. For all three genotypes,
isolated calli were then cultivated on the same selective
medium. After 9 weeks of culture on differentiation
medium (Tang et al., 2001) with 15 mg l±1 kanamycin,
adventitious shoots were observed (Fig. 2A). For the J-29
genotype, groups of between 1 and 10 transformed
adventitious shoots grew directly on primary explants.
About 40% of these adventitious shoots were converted
into plantlets.
Both frequency of zygotic embryos forming transgenic
calli and frequency of calli forming transgenic shoots was
in¯uenced by the genotypes of loblolly pine. The
frequency of putative transgenic calli ranged from 3.2%
 Genetic transformation of loblolly pine 839

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Fig. 2. Transgenic plants obtained with three genotypes of loblolly pine and evaluation of susceptibility to the Dendrolimus punctatus Walker and
Crypyothelea formosicola Staud. (A) Transgenic adventitious shoots derived from kanamycin-resistant calli (bar=1.2 cm). (B) Rooting of
transgenic shoot (root is indicated by arrow) (bar=1.5 cm). (C) Transgenic plantlet (on the right) and untransformed plant control (on the right)
obtained by in vitro culture, established in soil in the greenhouse for 5 months (bar=1.8 cm). (D) Loblolly pine trees located at Zhangjiajie
Loblolly Pine Orchard damaged by Dendrolimus punctatus Walker and Crypyothelea formosicola Staud (bar=0.0001 cm). (E) Eggs of
Dendrolimus punctatus Walker collected from loblolly pine trees located at Zhangjiajie Loblolly Pine Orchard (bar=0.3 cm). (F) Insect feeding
assay of transgenic plants from genotype J-29 that the CRY1Ac protein has been detected by Western blotting used for insect bioassays
(bar=0.5cm).

(genotype E-11) to 28.7% (genotype E-44) (data not
shown). Putative transgenic adventitious shoots (Fig. 2A)
were formed on the surface of kanamycin-resistant calli, 9
weeks after kanamycin-resistant calli were transferred to
differentiation medium. The frequency of adventitious
shoots ranged from 12.761.6% (genotype E-11) to
24.962.0% (genotype J-29) on the differentiation medium
supplemented with BA and IBA in the 9th week of culture
(Table 1). Root primordia were usually formed at the base
of shoots. Elongation and rooting of transgenic shoots
(Fig. 2B), and acclimatization of regenerated plantlets
were carried out according to a procedure previously
described (Tang et al., 2001). Rooting frequency
34.363.9% (genotype E-11) to 47.964.3% (genotype
J-29) (Table 1) was observed and both growth and
phenotype of regenerated plantlets appeared similar to
the untreated control. Regenerated plantlets from
transgenic organogenic calli of loblolly pine were trans-
ferred from 125 ml Erlenmeyer ¯asks into a perlite:peat-
moss:vermiculite (1:1:1 by vol) soil mixture, and
acclimatized plantlets were successfully established in
the soil in greenhouse (Fig. 2C).
According to the protocol described, more than 300
transgenic calli were obtained with J-29, more than 200
with E-44 and only 60 with the E-11 genotype. Between 5
and 20 shoots were generated from each of the 300 calli
obtained from the J-29. For E-44, 3±10 shoots were
obtained from each group of embryos from the 200
transgenic calli. Only 60 regenerated shoots were obtained
for the E-11 due to a low frequency of regeneration from
transformed calli. Molecular characterization of trans-
formed plants were performed on selected transgenic
plants from independent events obtained with all three
families.

Table 1. In¯uence of genotypes on the frequency (%) of organogenic calli forming kanamycin-resistant shoots and the frequency
(%) of transgenic shoots forming roots in loblolly pine
Experiments were repeated three times and each replicate consisted of 30 pieces of transgenic calli or 30 transgenic shoots. The frequency of
organogenic calli forming kanamycin-resistant shoots and the frequency of transgenic shoots forming roots were determined in the 9th week of
culture, respectively. Values represent the means 6SD.
Genotypes The frequency of calli forming The frequency of transgenic
transgenic shoots (%) shoots forming roots (%)

Transgene Control Transgene Control

E-11 12.761.6 13.461.5 34.363.9 35.664.8
E-44 16.761.9 17.862.9 34.565.7 41.264.7
J-29 24.962.0 26.362.7 47.964.3 51.665.7
840 Tang and Tian
Con®rmation of T-DNA integration and gene
expression
Polymerase chain reaction (PCR) was used for the primary
analysis of transformants. Loblolly pine genomic DNA
was extracted from fresh tissues of control and putative
transgenic plants, respectively. The primer set used for
ampli®cation of insert DNA was the nptII forward primer
(nfp) and reverse primer (nrp). PCR analysis was carried
out as a rapid identi®cation for the insert DNA in putative
transgenic plantlets derived from kanamycin-resistant calli
from three genotypes (E-11, E-44, and J-29) of loblolly
pine. The expected 717 bp band (for nptII) was ampli®ed
in the transformed plantlets, but not in the non-transformed
plant control (Fig. 3). These PCR results con®rm that the
regenerated plantlets from kanamycin-resistant calli con-
tain the transgene derived from the pB48.215 plasmid.
Southern hybridization of genomic DNA was then carried
out to con®rm that the T-DNA was integrated into the plant
genome.
Southern blotting was used to evaluate the number of T-
DNA copies integrated into the plant genome. Restriction
enzyme HindIII and probe (CRY1Ac) were chosen in
order to detect fragments of the T-DNA (Fig. 1). It was
postulated that the number of copies of the CRY1Ac gene,
located on the right side of the T-DNA, is in fact
representative of the copy number of the whole T-DNA.
Among the 50 plants studied, 35 plants (70%) presented
one T-DNA copy (Fig. 4), 8 (16%) two copies, 5 (10%)
three copies, but 1 (2%) plant also showed four copies and
1 (2%) ®ve copies (data not shown). These results are
consistent with observations made in various dicotyledo-
nous plants including tobacco, petunia, tomato, and
sun¯ower (Tinland, 1996; Zambryski, 1988). The integra-
tion of foreign genes in regenerated transgenic plants was
also observed in Pinus radiata via particle bombardment
(Walter et al., 1998). In this investigation, only transgenic
plants with one to three copies of the CRY1Ac were
Fig. 3. PCR analysis of DNA isolated from putative transgenic
plantlets of loblolly pine. Lane M: 1 kb DNA molecular markers
(Gibco-BRL); lane P: pB48.215 plasmid control; lane C: non-
transgenic regenerated plantlet control; lanes 1±6: transgenic
regenerated plants from genotypes E-22 (lanes 1, 2), E-44 (lanes 3, 4),
and J-29 (lanes 5, 6), respectively. The presence of the 717 bp band
ampli®ed from templates obtained from transgenic plantlets and
absence of the 717 bp band ampli®ed from templates obtained from
non-transformed plant control suggest that the T-DNA is integrated
into the pine genome.
selected for Northern hybridization, Western blot, and
insect bioassays.
Transgenic plantlets from independent transformation
events were analysed by Northern hybridization. Total
RNA was isolated from control and putative transgenic
plants using the procedure reported by Chang et al. (1993).
No bands were detected in non-transformed control plants,
whereas bands were observed in transgenic plants (Fig. 5).
These results con®rm that the foreign genes have been
integrated into the Pinus taeda genome and expressed at
the transcription level in transgenic plants.
Western blot hybridization was used to evaluate the
expression of gene at the protein level. Proteins were
extracted from about 1 g of fresh needles collected from
transgenic plants with positive Northern hybridization,
Downloaded from jxb.oxfordjournals.org at Osmania University on September 27, 2010
respectively, and the detection of the insecticidal protein
was performed by Western blotting (Sambrook et al.,
1989) using a rabbit polyclonal antiserum, raised against
the CRY1Ac protein previously puri®ed in the authors'
laboratories (dilution of 1/2000 v/v). A secondary goat
anti-rabbit antiserum alkaline phosphatase conjugate
(Sigma), was then used for ®nal detection, at a dilution
of 1/5000 (v/v). Expression of the CRY1Ac gene was
Fig. 4. Southern blot analysis of PCR-positive transformed plants.
DNA was prepared as described in the experimental protocol section.
Plasmid and genomic DNA were digested with the restriction enzyme
HindIII (Boehringer Mannheim) overnight at 37 °C, and was
hybridized (at 65 °C) with the 1.9 kb B.t. probe, corresponding to the
CRY1Ac gene (BamHI-SalI fragment of B.t. gene), which was
labelled with b-[32P]dCTP (Ready to Go Labeling Beads (Pharmacia)).
Lanes 1±4: DNA from transgenic plants of genotypes E-11, E-44, J-
27, and J-27, respectively (20 mg); lane P: plasmid DNA of pB48.215
(5 pg); lane C: DNA from non-transformed plant of genotype E-11
(20 mg).
Fig. 5. Northern blot analysis of total RNA from transgenic plants
and non-transformed plant control. Lane C: RNA from non-
transformed plant (15 mg); lanes 1±4: RNA from transgenic plants of
genotypes E-11, E-44, J-27, and J-27, respectively (15 mg). The
integrity and the amount of RNA applied to each lane were veri®ed by
ethidium bromide staining (low panel).

tested by Western analyses on proteins extracted from 48
transformed plantlets of the 50 independent transgenic
calli analysed in this investigation. In 42 of them, the
polyclonal antibody detected protein (Fig. 6). As shown in
Fig. 6, an expected signal of 68 kDa molecular weight was
observed with puri®ed CRY1Ac protein and with needle
extracts from transformed plants. However, in the other six
transformed plants, although the CRY1Ac gene was
detected by Southern blotting, the protein was not detected
by Western analyses. The absence of an immunosignal in
the six transformed plants was related to high copy
numbers and possible co-suppression. These six plants
were not resistant to the larvae.
Insect bioassay
Bioassays were performed using transgenic calli and plants
derived from all three loblolly pine genotypes. Insect
resistance of transgenic plants with introduced synthetic
Bacillus thuringiensis CRYIAc genes have been con®rmed
by insect bioassay (Cheng et al., 1998; Stewart et al., 1996;
Xiang et al., 2000). In this investigation, the transgenic
calli and regenerated plants con®rmed by Southern,
Northern, and Western blot analysis were selected for
insect bioassay. Insecticidal activity was evaluated by
placing larvae of Dendrolimus punctatus Walker and
Crypyothelea formosicola Staud on the transgenic calli
(Table 2) and transgenic plants to be tested (Table 3). The
eggs of Dendrolimus punctatus Walker and Crypyothelea
formosicola Staud were collected from loblolly pine trees
(Fig. 3D) at Zhangjiajie Loblolly Pine Orchard. The eggs
(Fig. 3E) were incubated according to the method
described in the Materials and methods. It was observed
that the larvae often bore small curves on the ®rst or second
day of feeding on the regenerated plants (Fig. 3F) posi-
tively identi®ed by Southern, Northern, and Western blot
analysis. Two days later, some larvae died and the others
ceased feeding and became stunted, depending on the age
of the larvae. However, the larvae on the non-transformed
control plants of the same age as the transformed ones and
susceptible plants continued to feed and grew well to more
Fig. 6. Western blot analysis of total proteins from fresh needles
separated on 10% SDS-PAGE and probed with rabbit anti-cry1Ac
antibody; 25 mg of total protein was used as template for transgenic
plants and negative control. Lane M: Standard protein molecular-
weight marker; lane C: negative control with an untransformed plant;
lanes 1±3: transformed plants containing cry1Ac protein; lane 4: pure
cry1Ac protein (0.5 mg).
Genetic transformation of loblolly pine 841
than 0.7 cm in length after 7 d of feeding. Eventually these
larvae pupated later without mortality, leaving behind
severely damaged plants.
Discussion
Through extensive evaluations of factors involved in the
particle bombardment-mediated transformation, the pre-
sent protocol was developed with which a large number of
stably transformed loblolly pine plants were generated.
This protocol, with slight modi®cation as necessary,
appears applicable to all loblolly pine families and other
coniferous species that have been recalcitrant to trans-
formation. This protocol could be useful to genetic
engineering of the commercially important loblolly pine
Downloaded from jxb.oxfordjournals.org at Osmania University on September 27, 2010
clones with genes conferring virus resistance, disease
resistance, stress tolerance, wood property, lignin contents,
and other economical qualities.
Mature zygotic embryos of loblolly pine were used as
target tissues for particle bombardment. Actively dividing
cells in embryos where the dividing state is established
during the 3 d pretreatment provided a window of
competence for transformation. Tobacco cells bombarded
at the M- and G2-phases have 4±6 times higher transform-
ation ef®ciencies than those at the S- and G1-phases (Iida
et al., 1991). T-DNA integrates preferentially into
sequences that can potentially be transcribed (Perlak
et al., 1991). Studies on the integration of T-DNA into
the plant genome strongly suggest that host DNA synthesis
is required (Gheysen et al., 1987). Thus, the greater the
number of actively dividing cells in the tissue being
bombarded, the higher probability of obtaining stable
expression in the bombarded tissue. In the present protocol
it was critical that the embryos were bombarded and were
cultured in TE medium for a time at least suf®cient to
induce cell division on the target tissue. Cells hit by the
DNA-coated particles at this stage were likely to be the
cells from which proliferating calli are derived. Ellis et al.
(1993) observed that the percentage of spruce somatic
embryos expressing the GUS gene after bombardment
increased progressively with advanced developmental
stages. The transformed calli developed on the surface of
the embryos. Among three different sizes of gold particles
(0.6, 1.0, and 1.1 mm), the smallest particles were most
ef®cient in delivering the genes. Different levels of helium
gas pressures (1,100, 1,350, and 1,550 pounds per square
inch) did not affect transformation ef®ciency (data not
shown).
The CRY1Ac protein was not detected by Western
analysis in six of the 48 transgenic plants where the
CRY1Ac gene was detected by Southern blotting. This
may be due either to a level of protein below the detection
threshold of the antibody, which is about 0.1% of total
protein in this assay, or to a lack of expression of the
protein. It could also be explained by a recombination
842 Tang and Tian
Table 2. Insect resistance analysis of transgenic callus from different genotypes of loblolly pine
Experiments were repeated three times and each replicate consisted of 30 insects. The controls were untransformed regenerants of the same age
as the transformed ones. insect mortality was determined on the 7th day of the insect bioassay. Values represent the means 6SD.
Genotypes Mortality of insect (%)

Dendrolimus punctatus Control Crypyothelea formosicola Control

E-11 58.664.6 8.261.1 69.164.3 8.960.8
E-44 54.963.7 7.761.3 67.765.6 8.361.1
J-29 62.764.3 9.161.2 76.866.7 9.761.3

Table 3. Insect resistance analysis of transgenic plants from different genotypes of loblolly pine
Experiments were repeated three times and each replicate consisted of 30 insects. The controls were untransformed regenerants of the same age
as the transformed ones. Insect mortality was determined on the 7th day of the insect bioassay. Values represent the means 6SD.

Downloaded from jxb.oxfordjournals.org at Osmania University on September 27, 2010

Genotypes Mortality of insect (%)

Dendrolimus punctatus Control Crypyothelea formosicola Control

E-11 68.365.5 7.261.3 69.164.3 9.161.8
E-44 64.764.7 8.761.7 68.763.6 8.761.3
J-29 72.166.4 9.561.6 75.866.7 9.861.6

event within the T-DNA that has altered the structure of
the CRY1Ac gene. As previously foreseen (Gould, 1998),
the CRY1Ac gene conferred resistance to Dendrolimus
punctatus Walker and Crypyothelea formosicola Staud, as
demonstrated by the bioassays. A correlation between the
detection of the insecticidal protein in needle extracts
from transformed plants and resistance to Dendrolimus
punctatus Walker and Crypyothelea formosicola Staud
was observed in most cases. However, the observations
suggested that, in some cases, the level of resistance and
development stage could be related. This aspect will be
checked carefully when the plants are transferred into soil
for a ®eld trial. Suitable insect management will be
implemented in parallel with monitoring the expression of
the CRY1Ac gene throughout the ®ve year experiment. Up
to now, all the results dealing with the evaluation of the
ef®ciency of a B. thuringiensis (B.t.) gene in agricultural
conditions have been obtained on annual crops (Huang
et al., 2002). Thus, the observations to be carried out on
loblolly pine will be very valuable for the future devel-
opment of a B.t. strategy on perennial trees. Besides the
stability of gene expression, it must be borne in mind that a
targeted insect can develop resistance to toxins from
B. thuringiensis (Rousch, 1997; Gould, 1998). Therefore,
the use of genes with different resistance mechanisms is
ideally required to maintain a B.t. strategy. The CRY1B
gene could be used in order to obtain a cumulative effect
with CRY1Ac (Guerreiro et al., 1998), since the gut
receptors are different for the two proteins. Furthermore,
integrated pest management, including trapping, using
parasitoids, nematodes or entomophagous fungi, and
adapted agricultural practices (optimizing of herbicides
and other pesticide uses), also have to be considered. This
is particularly relevant for a perennial tree like loblolly
pine.
The transformation of loblolly pine for resistance to
Dendrolimus punctatus Walker and Crypyothelea formo-
sicola Staud is a ®rst step in the process of creating insect-
resistant transgenic loblolly pine plants. Although eco-
nomically important, the Dendrolimus punctatus Walker
and Crypyothelea formosicola Staud is not restricted to
China and is currently present worldwide and considered
to be the most devastating and economically important
insect pest of loblolly pine. The practicality of this work
will bene®t not only the loblolly pine producers, but also
the environment worldwide. The next step could therefore
be the large-scale development of loblolly pine plants
resistant to Dendrolimus punctatus Walker and
Crypyothelea formosicola Staud in the ®eld. The same
approach could be applied to other loblolly pine families
and conifers. Genetically modi®ed plants from other
conifers could also be produced. The results presented in
this work open the way to new opportunities to improve the
loblolly pine species, not only for other agronomic traits
but also for those of technological interest.
Acknowledgements
The authors are grateful to Professor Zhigang Pan of the Chinese
Academy of Forestry Sciences for his constant support of this work
and excellent technical assistance, to Professor Weihua Zhong of
South China Agricultural University and Professor Dongxiang Xu of
Wulin University for the collection of seeds, to Professor Feng Guo
of Institute of Zoology, Chinese Academy of Sciences for technical
advice on insect breeding assays, to members of the Plant

Biotechnology Laboratory and the Biochemical Engineering
Laboratory of the Chinese Academy of Sciences for their helpful
collaboration during the bioassays. Part of this work was presented
in a laboratory meeting of the Forest Biotechnology Group, North
Carolina State University. We thank Professor Fan Ouyang, Dr Ron
Sederoff, and Dr Ross Whetten for their discussion and encourage-
ment and corrections of the text. This work was partially supported
by China High Technology Program `863' Project.
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