Transformation of Cabbage (Brassica oleracea L. var. capitata) with Bt cry1Ba3 Gene for Control of Diamondback Moth

Agricultural Sciences in China

November 2011

2011, 10(11): 1693-1700

Transformation of Cabbage (Brassica oleracea L. var. capitata) with Bt cry1Ba3 Gene for Control of Diamondback Moth YI Deng-xia, CUI Lei, LIU Yu-mei, ZHUANG Mu, ZHANG Yang-yong, FANG Zhi-yuan and YANG Li-mei Key Laboratory of Horticultural Crops Genetic Improvement, Ministry of Agriculture/Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

Abstract To obtain transgenic cabbage line with broad insect resistance, a new synthetic Bacillus thuringiensis cry1Ba3 gene was introduced into white cabbage via Agrobacterium tumefaciens-mediated transformation and 37 transformants were obtained. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that cry1Ba3 was successfully inserted into the genome of cabbage. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that cry1Ba3 was expressed. Western blot results confirmed the production of insecticidal protein encoded by cry1Ba3. Insect bioassays showed that transgenic cabbages effectively controlled both susceptible and Cry1Ac-resistant diamondback moth (DBM) larvae. Key words: Bacillus thuringiensis cry1Ba3, cabbage, diamondback moth, resistance

INTRODUCTION Cabbage (Brassica oleracea L. var. capitata) is one of the most important vegetables in the world. However, the cultivation of this vegetable is severely challenged by infestation of pests, including the major insect, Plutella xylostella (diamondback moth, DBM), which results in great loss of the yield and damage to the quality of cabbage production (Shelton et al. 1982; Gould et al. 1984; Theunissen et al. 1995). The conventional insect control method heavily relies on the intensive and extensive use of chemical pesticides. Chemical insecticides cause severe environmental pollution and bring about adverse effects on people and beneficial insects. Moreover, DBM have evolved resistance to chemical insecticides (Hama 1992; Shelton et al. 1993b). More recently, genetic engineering has provided a promising method for breeders to obtain insect resistant plants (Qaim et al. 2003). In 2009, 14 million farmers planted Received 30 January, 2011

134 million ha of transgenic crops in 25 countries (James 2009). The adoption of transgenic crops has saved pesticide costs and reduced environmental impact from pesticides (Qaim et al 2003; Kleter et al. 2007). Bt transgenic crops express insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt). Various Cry proteins are highly toxic to lepidopterans, coleopterans or dipterans, but do not harm people or the environment (Crickmore et al. 1998; Bravo et al. 2007). Although there are many Cry toxins, only a small number of Cry proteins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, etc.) have been used commercially in Bt crops (Bravo et al. 2008). Agrobacterium tumefaciens-mediated transformation of cabbage has been reported with some Cry toxins such as Cry1Ab, Cry1Ac, and Cry1C. Bioassays of Bt cabbage plants showed that DBM was effectively controlled by these Bt proteins (Metz et al. 1995; Jin et al. 2000; Bhattacharya et al. 2002). Insect resistance genes are very limited in availability for plant breeders. Hence, novel Bt genes will be new resource

Accepted 3 June, 2011

Correspondence YANG Li-mei, Tel: +86-10-82108756, Fax: +86-10-62174123, E-mail: [email protected]

© 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60167-3

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for the transformation of plants. A cry1Ba3 gene obtained from the Bt strain UV17 was recently cloned by Wang et al. (2008). Its predicted molecular weight is 140 kD. Bioassays showed that the Cry1Ba3 protein possessed distinct insecticidal activities against DBM, and there was no cross resistance with Cry1Ac (Liu et al. 2010). Cry1Ba3 will be available for use in gene pyramiding to inhibit development of resistant insects. The objective of this study was to obtain transgenic cabbage expressing cry1Ba3 via A. tumefaciens-mediated transformation. Deployment of the resulting plants may expand the insect resistance gene spectrum for cabbage pests.

MATERIALS AND METHODS Plant materials Cabbage inbred line (B. oleracea L. var. capitata) CA213 was used as transformation material.

Diamondback moths Susceptible and Cry1Ac-resistant DBM were reared on cabbage plants in an environmental chamber at (27±1)°C, (35±2)% RH, and photoperiod of 16 h/8 h (light/dark). The DBM colonies were started with material collected

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from America in 2005 and since then reared continuously in the Laboratory of Entomology, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. Newly molted second instar larvae were used for insect bioassays. At the time of the bioassay, the Cry1Ac-resistant population was over 1 000-fold more resistant to Cry1Ac protoxin than the susceptible one.

Binary vector The A. tumefaciens strain EHA105 harboring a binary vector pCSBaN with the synthetic cry1Ba3 and the neomycin phosphotransferase-II (npt II) genes was used for cabbage transformation. The cry1Ba3 and npt II genes were both regulated by the enhanced cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. The npt II gene was a selectable marker gene allowing the transformed shoots to grow in media containing kanamycin. The cry1Ba3 gene fragment was introduced into the expression vector pCAMBIA2300, which carried the npt II gene, and then transformed into Escherichia coli strain JM110. The resulting plasmid was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation (Mersereau et al. 1990). The T-DNA region of the binary vector pCSBaN is shown in Fig. 1.

Fig. 1 The binary vector pCSBaN used in cabbage transformation. LB is T-DNA left border, RB is T-DNA right border, eCaMV35 is enhanced CaMV35S promoter, Tnos is nopaline synthase terminator. The Hind III sites were used for DNA digestion in Southern blot. A 1.222 kb PCR fragment containing part of the cry1Ba3 gene was used as a probe for DNA hybridization.

Plant regeneration Various factors that might influence shoot regeneration were tested with non-inoculated explants. Tested parameters included types of explant (hypocotyl and cotyledon), age of explant (3, 5, 7, and 9 d), and

concentration of 6-BA (1, 2, and 3 mg L-1) in combination with NAA ( 0.01, 0.05, and 0.1 mg L-1). Explants were cut and placed on MS medium (Murashige et al. 1962). All treatments were carried out in triplicate. Regeneration frequency of buds was scored after 30 d.

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Transformation of Cabbage (Brassica oleracea L. var. capitata) with Bt cry1Ba3 Gene for Control of Diamondback Moth

Transformation procedure The transformation was following the procedures of Cao et al. (2008) and Metz et al. (1995). Full-strength MS salts and 28 g L-1 sucrose were present in all the following media except in Luria-Bertani (LB) medium. Seed germination and pre-culture: Cabbage seeds were surface-sterilized in 70% ethanol for 2 min and then in 10% Clorox for 10 min. The seeds were rinsed four times with sterile water and placed on hormone-free MS medium for germination at 25°C under 16 h/8 h light/dark regime. 5-d-old hypocotyls were cut into 10 mm segments and placed on MS medium. They were cultured at 25°C under 16 h/8 h light/dark regime for 2 d. Agrobacterium cells derived from a single colony harboring pCSBaN were suspended in LB medium containing 100 mg L-1 kanamycin and cultured at 28°C with shaking at 250 r/min until the OD600 reached to 0.6-0.9. Pre-cultured explants were immersed in the Agrobacterium suspension for 10 min and then rinsed once with MS liquid medium. Inoculated explants were placed on MS medium containing 2 mg L -1 6benzylaminopurine (6-BA) and 0.1 mg L -1 αnaphthaleneacetic acid (NAA). They were incubated at 28°C in the dark for 3 d. Explants were then transferred to regeneration medium supplemented with 150 mg L-1 timentin, 2 mg L-1 6-BA, and 0.1 mg L-1 NAA and cultured at 25°C under 16 h/8 h light/dark regime to eliminate Agrobacterium. 7 d later, explants were transferred to selective medium that contained 150 mg L-1 timentin, 20 mg L-1 kanamycin, 2 mg L-1 6-BA, and 0.1 mg L-1 NAA. Resistant plantlets emerged 5 wk later. The green plantlets were excised from the calli and put into MS medium that had a higher concentration of kanamycin (25 mg L-1) to reduce the number of escapes. Plantlets that remained green were placed into rooting medium containing 0.1 mg L-1 NAA and 0.1 mg L-1 3indolebutyric acid. Rooted plants were transplanted into soil.

Molecular analysis PCR and Southern blot analyses Genomic DNA was extracted from fresh leaves of putative transformants and non-transformed plants by the CTAB method (Doyle et al. 1987). Specific forward and reverse primers

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were designed for PCR analysis. The sequences of the primers for cry1Ba3 were 5´-CCAGAGACTA CTCCGACTATTGCG-3´ (forward) and 5´-TACA CTTCCCCATTGCCACTAA-3´ (reverse). PCR was carried out for cry1Ba3 in 20 μL in a thermal cycler with the following amplification program: 94°C for 4 min, 1 cycle; 94°C for 60 s, 60°C for 60 s, 72°C for 90 s, for 30 cycles; 72°C for 10 min, 1 cycle. The amplified products of cry1Ba3 were electrophoresed in 1% agarose gels in 0.5×TBE buffer. For Southern blot analysis, 15 μg genomic DNA per sample was digested with Hind III (there are many Hind III restriction sites in the cabbage genome). The digested DNA was electrophoresed in 1% agarose gel and transferred onto Hybond N+ nylon membrane. The DNA probe was prepared from a PCR-amplified 1.222 kb fragment covering the part of the cry1Ba3 gene. Blots were obtained following the manufacturer’s instruction with the DIG High Prime DNA Labeling and Detection Kit II from Roche Molecular Biochemicals (Basel, Switzerland). After hybridization, the membrane was washed sequentially with stringency solutions to blocking solution. Finally, the membrane was washed with colour substrate solution (NBT/BCIP mixture) for colour detection. RT-PCR analysis Total RNA was isolated from fresh leaves of transformed plants and non-transformed plants using a Total RNA Extraction Kit from Biomed-tech company (Beijing, China) and the manufacturer’s protocol. Synthesis of first-strand cDNA was performed with Revert AIDTM First Strand cDNA Synthesis Kit from Fermentas company (Burlington, Ontario, Canada), and then PCR analysis was carried out using the cry1Ba3 specific forward and reverse primers for the detection of transcripts using procedures outlined previously. β-actin was used as an internal reference. The PCR products of cry1Ba3 were electrophoresed in 1% agarose gels in 0.5×TBE buffer. Western blot analysis Total protein was extracted from fresh leaves of transformed and non-transformed plants with a protein extraction buffer as described by Sambrook et al. (1989). The protein extraction buffer contained 200 mmol L-1 Tris·HCl (PH 8.0), 100 mmol L-1 NaCl, 400 mmol L -1 sucrose, 14 mmol L -1 βmercaptoethanol, 1 mmol L-1 phenylmethyls ulfonyl fluoride and 0.05% Tween-20. After grinding, the mixture

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was centrifuged at 12 500 r/min for 20 min at 4°C. Protein concentrations in the supernatant were determined using a Lowry protein assay (Larson et al. 1986). The supernatant was subjected to electrophoresis in 10% SDS-polyacrylamide gels as described by Sambrook et al. (1989). Then protein was electrotransferred onto polyvinylidene-fluoride membrane. Western blot analysis was carried out according to the enhanced chemiluminescence Western blotting protocol described by Bradd and Dunn (1993). The primary antibody was raised in rabbit against proteins isolated from Bt. This antibody had the reactivity to Cry1Ba3 protein. It was diluted at 1:500 for probing. Secondary antibody against rabbit IgG (whole molecule) was produced in goat and purchased from Sigma company (Saint Louis, Missouri, USA).

Insect bioassay For detached leaf bioassays a leaf of each cry1Ba3 plant and non-transformed cabbage was cut into two sections, each 50 mm in diameter. One section was used for bioassay of susceptible DBM larvae while the other was used for bioassay of Cry1Ac-resistant DBM larvae. Each leaf section was placed in a 90-mm Petri dish (a moist filter paper was put on the bottom of the Petri dish beforehand to maintain moisture). Ten 2nd instar DBM larvae were placed on the surface of each leaf section, and then the Petri dishes were sealed with Parafilm. All insect bioassays were carried out in triplicate. Leaf damage (visual estimate) and mortality of larvae were scored after 6 d.

RESULTS Plant regeneration and transformation Plant regeneration Effects of seedling age, types of explant, and hormones on shoot regeneration were tested to facilitate transformation of cabbage. Based on these results, the hypocotyls from 5-d-old seedlings and the combination of 2 mg L-1 6-BA with 0.1 mg L-1 NAA were used for subsequent transformation experiments. Selection of transformants Kanamycin-resistant plantlets emerged 5 wk later after transformation. The

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majority of the initially green shoots gradually turned white or brown and some became necrotic after subsequent subcultures. After rooting healthy plants were transplanted into soil. A total of 37 kanamycin-resistant plantlets were recovered of which 29 survived. The transgenic plants were morphologically normal.

Molecular analysis of transgenic plants PCR and Southern blot analyses Kanamycin-resistant plants were subjected to PCR analyses to confirm the presence of the cry1Ba3 gene. The expected 1.222 kb band representing the cry1Ba3 fragment was amplified from the genomic DNA of transgenic plants and the pCSBaN plasmid. No band was amplified from the genomic DNA of non-transformed plants. Representative results of the PCR analysis are shown in Fig. 2. Lane 5 shows a kanamycin-resistant plant in which the cry1Ba3 gene was not integrated.

Fig. 2 PCR analysis of genomic DNA from kanamycin-resistant plants. Lane 1, 2 kb molecular weight marker; lane 2, pCSBaN carrying cry1Ba3 gene; lane 3, blank lane; lane 4, non-transformed cabbage; lanes 5-9, kanamycin-resistant plants.

Southern blot analysis provided additional evidence of integration of cry1Ba3 into the cabbage genome. DNA isolated from the leaves of PCR-positive plants and nontransformed plants was digested with Hind III and hybridized with the probe from the PCR products of cry1Ba3 gene. Most of the transformed plants showed hybridization signals to the probe. No signal was detected for the non-transgenic plants. Representative results of the Southern blot are shown in Fig. 3. There was a single insertion site in some transgenic plants while others possessed two or more insertion sites. RT-PCR analysis Transgenic cabbages confirmed by PCR and Southern blot were analyzed by RT-PCR to evaluate the presence of cry1Ba3 transcripts. For all the transgenic cabbages, RT-PCR amplified the expected 1.222 kb cry1Ba3 band and the 0.196 kb band for the β-actin internal reference. Only the 0.196 kb internal

© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.

Transformation of Cabbage (Brassica oleracea L. var. capitata) with Bt cry1Ba3 Gene for Control of Diamondback Moth

reference band was amplified from non-transformed cabbage (Fig. 4). Expression of cry1Ba3 Western blots were performed on the RT-PCR-positive plants to identify the production of insecticidal crystal protein encoded by cry1Ba3. 100 μg of total soluble protein extracted from transformed and non-transformed cabbage was subjected to Western blot analysis. The results demonstrated the presence of the expected 140 kD Cry1Ba3 protein in some transformed cabbages while non-transformed plants did not express the protein. Some transformed plants did not express the expected protein though they were confirmed by RT-PCR (data not shown). Representative results are shown in Fig. 5.

Insect bioassay Transgenic plants expressing Cry1Ba3 protein were

Fig. 3 Southern blot analysis of PCR-positive plants. Lane 1, plasmid pCSBaN; lane 2 , non-transformed cabbage; lanes 3-11, transformed cabbages.

Fig. 4 RT-PCR analysis of transformed cabbages. Lane 1, 2 kb molecular weight marker; lane 2, is pCSBaN carrying the cry1Ba3 gene; lane 3, non-transformed cabbage; lanes 4-7, transformed cabbages.

Fig. 5 Western blot analysis of transformed cabbages. Lane 1, protein marker; lane 2 , Cry1Ba3 protein; lane 3, non-transformed cabbage; lanes 4-6 are transformed cabbages.

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tested for insect resistance by exposing detached leaves individually to susceptible and Cry1Ac-resistant DBM. The results showed that transgenic plants were resistant to susceptible DBM larvae and could also control DBM larvae resistant to Cry1Ac toxin (Table). There was no mortality on untransformed plants. Some transgenic plants showed no leaf damage and caused complete mortality of larvae within 2 to 3 d. After infestation, leaf disks from control cabbages were completely eaten, and live larvae were present (Fig. 6).

DISCUSSION The kind and age of explant, concentration of 6-BA in combination with NAA, length of inoculation with Agrobacterium, length of pre-cultivation and cocultivation, usage of AgNO 3 , concentration of kanamycin, and some other parameters have been reported to influence transformation of cabbage (Metz et al. 1995; Pius et al. 2000; Rafat et al. 2010). In our study, various factors that might influence shoot regeneration were tested to obtain transgenic cabbage. We mainly focused on the effects of 6-BA and NAA on regeneration and transformation because the combination of 6-BA with NAA has been reported to increase the frequency of Brassica shoot regeneration (Metz et al. 1995; Paul et al. 1999; Cao et al. 2008). Western blots indicated that some plants did not express Cry1Ba3 protein although the presence of the gene in these plants was confirmed by PCR and Southern blot. The cause of this lack of expression is unknown. It has been reported that gene silencing occurs at transcriptional and post-transcriptional levels due to introduction of two or more similar genes, promoters or enhancers into a plant genome (Meyer 1998; Matzke et al. 2000). The synthetic cry1Ba3 gene was modified to change the codon usage to fit plant codon preference, and the gene was controlled by the enhanced cauliflower mosaic virus 35S promoter for efficient transcription. The expression of cry1Ba3 gene in cabbage is worthy of further study. CA21-3 is a cabbage inbred line that possesses the features of superior quality and high yield. Molecular analyses proved that cry1Ba3 gene has been inserted into the genome of CA21-3. Insect assays demonstrated that some Cry1Ba3 plants could cause complete mor-

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Table The results of detached leaf bioassay of transgenic cabbages after 6 d Plant Control YA-1 YA-2 YA-3 YA-4

Mortality (%) 0b 100 a 96.77 a 93.33 a 100 a

Susceptible DBM Estimated defoliation (%) 90-95 0-1 0-1 1-2 0-1

Mortality (%)

Cry1Ac-resistant DBM Estimated defoliation (%)

0b 100 a 100 a 96.77 a 96.77 a

90-95 0-1 0-1 0-1 0-1

Different letters following mortality values indicate statistically significant difference based on the Duncan’s new multiple range test at P<0.05.

Fig. 6 In vitro bioassay of non-trangenic and transgenic cabbage with DBM. A, assays with DBM susceptible to Cry1Ac. B, assays with DBM resistant to Cry1Ac. 1, non-transformed control; 2-6, transgenic plants.

tality of susceptible and Cry1Ac-resistant DBM. Homozygous insect-resistant plant lines can be obtained by self-pollination, test-crossing, and molecular analyses. Such lines may be useful for insect resistance breeding in the future. DBM has evolved resistance to Cry1Ab, Cry1Ac and some other Bt toxins in the field (Shelton et al. 1993a; Perez et al. 1996; Tang 1997; Bravo et al. 2008). Thus, Bt crops are threatened by the evolution of insect resistance. Various strategies have therefore been suggested to delay insect resistance evolution: (I) high dose plus refuge strategy; (II) use of two or more insectresistance genes; (III) deployment of transgenic plants expressing toxin genes at a moderate level so that not all susceptible insects are killed; (IV) transgenic plants expressing toxin in certain organs, during a certain period or in response to an environment signal. A high dose of a single Bt gene in conjunction with refuges of non-Bt plants has been applied in several countries (Gould 1998; Tabashnik et al. 2003, 2009). Pyramiding two or more Bt toxins in plants is a promising way to postpone insect resistance evolution (Zhao et al. 2003;

Bates et al. 2005; Halpin 2005; Tabashnik et al. 2008, 2009). Cui et al. (2009) obtained Bt cry1Ia8 cabbage, and the results of bioassays showed that cry1Ia8 plants were resistant to the larvae of both susceptible and Cry1Ac-resistant DBM. Cry1Ba3 was successfully inserted into the genome of cabbage in this research. Therefore, cry1Ia8 and cry1Ba3 genes can be pyramided by sexual crosses or by sequential transformation. Deployment of resulting plants may inhibit the evolution of insect resistance.

Acknowledgements We thank Prof. Huang Dafang, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, China, Dr. Zhang Jie,Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China, and Dr. Lang Zhihong, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China, for their help in providing gene and vector. We are grateful to Dr. Zhang Youjun, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China. for test larvae supply

© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.

Transformation of Cabbage (Brassica oleracea L. var. capitata) with Bt cry1Ba3 Gene for Control of Diamondback Moth

and help in bioassays. Thanks are due to Prof. Elizabeth Earle, Department of Plant Breeding and genetics, Cornell University, USA, and Dr. Cao Jun, Athenix Corporation, USA, for providing the Brassica genetic transformation protocol and constructive advice on writing the manuscript. This work was supported by the grants from the National High Technology Research and Development Program of China (863 Program, 2008AA10Z155), the National Natural Science Foundation of China (31071697), and the earmarked fund for the Modern Agro-Industry Technology Research System, China (nycytx-35-gw01).

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