and Technology, Clear Water Bay, Kowloon, Hong Kong,. China. M.C. Ma. Department of Entomology, University of Maryland,. College Park, MD 20742â5141, ...
Plant Cell Reports (2000) 19 : 251–256
Q Springer-Verlag 2000
Y. Xiang 7 W.-K.R. Wong 7 M.C. Ma R.S.C. Wong
Agrobacterium-mediated transformation of Brassica campestris ssp. Parachinensis with synthetic Bacillus thuringiensis cry1Ab and cry1Ac genes
Received: 22 February 1999 / Revision received: 14 April 1999 / Accepted: 26 April 1999
Abstract An effective plant regeneration procedure and a gene transfer system via Agrobacterium tumefaciens were developed in Brassica campestris ssp. parachinensis. Hypocotyls from 5-day-old seedlings with 2 days pre-culture were infected with Agrobacterium strain MOG301 harboring a binary vector containing a synthetic Bacillus thuringiensis (B.t.) cry1Ab or cry1Ac gene with full codon-modification. After culture and selection on MS medium supplemented with 4.0 mg/l BAP, 2.0 mg/l NAA, 70 mM AgNO3 and 50 mg/l kanamycin, a number of kanamycin-resistant plantlets were regenerated. PCR and Southern blotting analysis were used to identify and characterize the transgenic plants with the integrated cry1Ab or cry1Ac gene. Western blotting analysis of the transgenic plants confirmed the expression of insecticidal proteins encoded by cry1Ab or cry1Ac. Subsequent bioassay with larvae of the Diamondback moth, Plutella xylostella, demonstrated that the transgenic plants were resistant to feeding damage. Key words Agrobacterium-mediated transformation 7 B.t. insecticidal protein gene 7 B. campestris ssp.parachinensis 7 Transgenic plants 7 Insect resistance
Introduction Chinese flowering cabbage (Brassica campestris ssp. parachinensis) is one of the most important vegetables Communicated by C.F. Quiros Y. Xiang 7 W.-K.R. Wong 7 R.S.C. Wong (Y) Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China M.C. Ma Department of Entomology, University of Maryland, College Park, MD 20742–5141, USA
in southern China. Like many other crop plants, the production of this vegetable is challenged by many stresses including infestation of insects. The conventional insect control method is mainly dependent on the intensive and extensive use of chemical pesticides, which have drawbacks such as damage to the ecological system and residual poisoning of humans and animals. Therefore, it is desirable to develop insect-resistant plants through the introduction of foreign insecticidal genes. Among the insecticidal genes, Bacillus thuringiensis (B.t.) crystal protein (or d-endotoxin) genes have been proven effective in controlling insect larvae in many crop plants (Perlak et al. 1990, 1993; Koziel et al. 1993; Cheng 1998). Recently, a report on the transformation of a B.t. insecticidal protein gene to B. oleracea was published (Metz et al. 1995). However, applications to other Brassica leafy vegetable species are worth pursuing. Efficient gene transfer and plant regeneration systems are necessary for the development of transgenic plants. Various explants have been successfully used for Agrobacterium-mediated transformation of several Brassica species (Radke et al. 1988; De Block 1988; Swanson and Erickson 1989; Barfield and Pua 1991). In contrast to these successfully transformed Brassica species, B. campestris is known to be recalcitrant to shoot regeneration (Jain et al. 1988; Narashimhulu and Chopra 1988). In the past few years, several studies have reported the genetic transformation and plant regeneration of oilseed Brassica campestris (Mukhopadhyay et al. 1992) and the vegetable B. campestris ssp. pekinensis (Jun et al. 1995). However, a low regeneration frequency and very few transformants were obtained. As to B. campestris ssp. parachinensis, efficient plant regeneration and the production of transgenic plants have not yet been established. In this report, we describe an efficient method of plant regeneration from hypocotyls of Brassica campestris ssp. parachinensis and a procedure of Agrobacterium-mediated transformation in the development of insect-resistant plants. As diamondback moth is the
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major pest for this leafy vegetable synthetic B.t. cry1Ab and cry1Ac genes, encoding toxin proteins specific for controlling this pest, were used.
Materials and methods Plant materials and preparations Five varieties of Brassica campestris ssp. parachinensis with different dates of maturity (BP-40, BP-50, BP-60, BP-70 and BP80) were obtained from a local seed supplier. Seeds were submerged in 70% ethanol for 5 min and then in 30% CLORO TM bleach solution (about 2% sodium hypochlorite) for 45 min. These seeds were then rinsed three times in sterilized ddH2O and germinated in light on 1/2-strength MS medium (Murashige and Skoog 1962) without hormones for 5 days. After germination, the hypocotyls were cut into 7- to 10-mm segments and placed on culture medium. Shoot regeneration To optimize the culture medium for shoot regeneration, we cultured hypocotyls excised from 5-day-old seedlings on media with different concentrations of 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA): in basal MS medium supplemented with 40 mM AgNO3, three concentrations of BAP (2, 4, or 6 mg/l) and NAA (1, 2 and 3 mg/l) were tested. Construction of binary vectors and Agrobacterium strain The 1.845-kb cry1Ab (Perlak et al. 1991) and cry1Ac (Perlak et al. 1990) genes were constructed by a single-step polymerase chain reaction (PCR) assembly of the chemically synthesized oligonucleotides from sections of the gene sequence and cloned into pGEM3Zf (c) vector to produce pCryIAb or pCryIAc (Xiang et al. 1997). The gene fragments cut by BamHI and NaeI from pCryIAb or pCryIAc were inserted in pBI121 to produce pBICryIAb or pBI-CryIAc. A 2.8-kb EcoRI-HindIII fragment, containing the CaMV 35S promotor, cry1Ab or cry1Ac and nosterminator, was cut from pBI-CryIAb or pBI-CryIAc and inserted into plasmid pCAMBIA2301. This is a binary vector obtained from the Center for the Application of Molecular Biology to International Agriculture in Canberra, Australia. The T-DNA regions of the resulting binary vectors, pKM-CryIAb and pKM-CryIAc, are presented in Fig. 1. The two binary vectors were then separately introduced into Agrobacterium strain MOG301 (Hood et al. 1993).
medium (Chilton et al. 1974) were pelleted, washed and then resuspended in PCM to an O.D600 value of 0.1–0.2 before use. The pre-cultured hypocotyls were immersed in the Agrobacterium suspension for 5–10 min, blotted dry with sterile filter paper to remove excess bacteria and co-cultivated on PCM medium at 25 7C for 2 days. The infected hypocotyls were washed with sterile ddH2O and then transferred onto low selective medium (LSM) consisting of MS medium with 1.0 mg/l BAP, 0.1 mg/l NAA, 30 mg/l kanamycin, 70 mM AgNO3 and 500 mg/l carbencillin. After culture for 4 days, the hypocotyls with calli were transferred onto selection and regeneration medium (SRM) containing MS medium with 4.0 mg/l BAP, 2.0 mg/l NAA, 50 mg/l kanamycin, 70 mM AgNO3 and 500 mg/l carbencillin, for further selection and shoot regeneration. After 3 weeks, the hypocotyls were then subcultured once on SRM. When green shoots were regenerated, they were transferred to rooting medium (RTM), containing 1/2-strength MS with 0.1 mg/l NAA, 60 mg/l kanamycin and 500 mg/l carbencillin, for root growth and kanamycin selection. Resulting plantlets were transferred to soil in potts. PCR amplification Genomic DNA was prepared from leaf tissue of the putative transgenic plants. PCR of the genomic DNA was carried out to identify the presence of the B.t. gene with gene-specific primers. For cry1Ab, two primers, 5’GGACA ACAAC CCAAA CATCA ACG-3’, located at position 3–25, and 5’TTCGG CAGGG CACAA ACTCA ATAC3’, at position 1802–1824, were used. The amplified DNA fragment from the PCR was 1821 bp. For cry1Ac, two primers, 5’TACTT GGTGG AGAAC GCATT G3’ and 5’AGTCG CTGGA TTGGA GATTG3’, were used to amplify a 1640-bp fragment. PCR was performed in a thermal cycler withthe following amplification program: 1 cycle of 3 min at 94 7C, 2 min at 50 7C and 2 min at 72 7C; 30 cycles of 1 min at 94 7C, 1 min at 50 7C and 1 min at 72 7C; a final cycle of 72 7C for 7 min. After PCR, the resulting samples were subjected to electrophoresis using a 1.0% agarose gel. Southern blot analysis Genomic DNA was digested with BamHI for Southern hybridization to confirm the integration of cryIAb or cryIAc into the plant genome. The digested genomic DNAs were fractionated on 0.7% agarose gels, transferred onto a nylon membrane and hybridized to digoxigenin (DIG)-labeled probes according to the supplier’s instructions (Boehringer Mannheim). The DNA probes were prepared by labeling the 1.845-kb cry1Ab or cry1Ac gene fragment using a random primed labeling method. The two probes contain a common region to both cry1Ab and cry1Ac of 1.351 kb.
Transformation Western blot analysis Hypocotyls were cultured for 2 days on pre-culture medium (PCM) containing MS medium with 1 mg/l BAP and 0.1 mg/l NAA. Overnight cultures of Agrobacterium in AB-minimal
Fig. 1 T-DNA regions ofthe binary vectors pKM-cryIAb and pKM-cryIAc. LB Left border, RB right border, NPTII neomycin phosphotransferae gene, P35S CaMV 35S promoter, NT 3’termination signal of nopaline synthase
The regenerated plants from transformed hypocotyls were analyzed by Western blotting to identify the expressed insecticidal proteins encoded by cry1Ab or cry1Ac. Protein was extracted from individual transgenic plants by grinding mature leaf tissue in a buffer containing 50 mM Na2CO3, pH 9.5, 100 mM NaCl, 0.05% Triton X-100, 0.05% Tween-20, 2.0 mM phenylmethylsulfonyl fluoride and 1.0 mM leupeptin. The ground mixture was centrifuged at 12,000 g for 15 min. The supernatant was subjected to gel electrophoresis for Western blot analysis
253 following the procedure of Ausubel et al. (1994). The polyclonal antiserum was prepared in rabbit according to standard procedures using proteins isolated from B t. kurstaki. This antiserum has cross reactivity to both Cry1Ab and Cry1Ac proteins.
reported by Mukhopadhyay et al. (1992) that a high concentration of silver nitrate (70–90 mM) is beneficial to plant regeneration for Agrobacterium-transformed hypocotyls.
Insect bioassay All positive transgenic plants expressing the CryIAb or CryIAc proteins were tested for lethality and growth retardation of diamondback moth (Plutella xylostella) larvae using a whole plant-feeding assay. The test plants were individually infested with 31–63 larvae of 3–5 mm in length for 7 days at 20 7C under 16/8-h light/dark regime. The test plants were classified into three groups, i.e. highly resistant, moderately resistant and susceptible, based on larval response and severity of damage to leaf tissue.
Results Plant regeneration As a first step, the shoot regeneration procedure from hypocotyls was established for transformation. Five varieties of Brassica campestris ssp. parachinensis, BP40, BP-50, BP-60, BP-70 and BP-80, were tested. All media supplemented with a combination of BAP and NAA at different concentrations induced callusing with varying results. The best result of 37.9% was obtained from the combination of 4.0 mg/l BAP and 2.0 mg/l NAA (Table 1). About 40% shoot regeneration was obtained from hypocotyls of 5-day-old seedlings, while explants from seedlings younger and older than 5 days resulted in 26.4% and 17.1% shoot regeneration, respectively. The addition of at least 40 mM silver nitrate to the regeneration medium was appropriate and essential for shoot formation. Shoots were regenerated via organogenesis from the callus cells around the end of hypocotyls after about 5 weeks of culture. Based on these results, the use of hypocotyls from 5-day-old seedlings and a plant hormone combination of 4.0 mg/l BAP and 2.0 mg/l NAA was adopted for subsequent transformation. However, the concentration of silver nitrate was increased to 70 mM in transformation experiments with Agrobacterium. It has been
Transformation and selection of transformants The 2-day precultured hypocotyls from 5-day-old seedlings were co-cultivated with Agrobacterium tumefaciens for 2 days. After co-cultivation, the infected hypocotyls were placed on LSM medium with low selection pressure. Under these conditions, the ends of the hypocotyls gradually initiated the formation of callus. Some of these calli grew more quickly than others. After 4 days of culture on LSM medium, all hypocotyls were transferred onto the SRM medium for further selection with a higher concentration of kanamycin and subsequent shoot regeneration. During the process of selection, the successfully transformed hypocotyls continued to grow vigorously to produce calli, whereas the untransformed ones failed to form callus and eventually bleached and became necrotic within 3 weeks. Shoots were usually regenerated within 4–6 weeks on the SRM medium after co-cultivation. Some cultured hypocotyls formed large calli 1.2–1.5 cm in diameter but failed to regenerate shoots. Only those hypocotyls producing small calli 0.6–1.0 cm in diameter easily regenerated shoots for further development. During the selection culture, subculturing the explants with a change of fresh medium containing 50 mg/l kanamycin greatly reduced the number of escapes. Leaves often appeared first at the initial stage of shoot regeneration before the complete shoots were formed, suggesting that shoots were regenerated via organogenesis. The average shoot regeneration percentage for the five Brassica vegetable varieties after Agrobacterium infection and selection was 5.04%, with a total of 119 shoots produced (Table 2). It was greatly reduced when compared to the 37.9% shoot regeneration from culture without the infection of Agrobacterium and selection with kanamycin (Table 1).
Table 1 Percentage of hypocotyls of five varieties of Brassica campestris ssp . parachinensis with shoot regeneration on media with various concentrations of BAP and NAA Hypocotyls with shoot regeneration (%) Combination (mg/l) BAP 2.0 BAP 4.0 BAP 6.0 Average
cNAA cNAA cNAA cNAA cNAA cNAA cNAA cNAA cNAA 17.9
1.0 2.0 3.0 1.0 2.0 3.0 1.0 2.0 3.0
BP-40
BP-50
BP-60
BP-70
BP-80
Average
17.5 14.7 16.3 25.0 30.4 21.1 20.5 12.6 2.8 22.5
23.5 18.1 20.0 29.5 36.8 23.2 28.3 17.5 5.6 14.2
16.0 14.5 13.6 16.2 26.9 10.0 19.5 9.8 1.5 25.1
22.7 17.0 23.7 33.7 47.5 25.4 30.1 19.7 6.5 25.7
25.3 16.9 19.9 37.1 47.9 26.2 29.3 20.5 8.2 21.1
21.0 16.2 18.7 28.3 37.9 21.2 25.5 16.0 4.9
254 Table 2 Kanamycin-resistant plants from Agrobacterium-infected hypocotyls of Brassica campestris ssp . parachinensis Varieties
BP-40 BP-50 BP-60 BP-70 BP-80 Total a
Number of explants
380 561 286 463 671 2362
Km r transformants a
Plants regenerated Number
Percentage
Number
Percentage
14 16 8 43 38 119
2.89 2.50 2.10 8.00 4.77 5.04 b
8 11 5 33 29 86
2.11 1.98 1.75 7.13 4.32 3.64 b
Kanamycin-resistant transformants Average percentage
b
When the shoots were completely formed and had grown to about 3 cm in length, they were separated from the callus and transferred onto RTM medium with 60 mg/l kanamycin. This step was used to further select the successfully transformed shoots. By the end, 86 plantlets were obtained. The average percentage of kanamycin-resistant regenerants was 3.64% (Table 2). After the plantlets were transferred to potting soil, 70 plants survived and grew to maturity. Confirmation of T-DNA integration To confirm the presence of the cry1Ab or cry1Ac gene in the regenerated plants, we subjected all R0 plants to PCR analysis with the primers specific for the synthetic cry1Ab or cry1Ac genes. The results indicated that DNA from 65 plants (92.9%) could be amplified to produce the specific DNA band, i.e. 1.8 kb for the cry1Ab-transformed plants and 1.64 kb for the cry1Actransformed plants. Representative results of the PCR analysis are shown in Fig. 2A and B. The successfully regenerated plants confirmed by PCR were further analyzed by Southern blotting hybridization. Genomic DNA prepared from the regenerated plants was digested with BamHI, as there is a unique site in the T-DNA region of the binary vectors pKM-cryIAb and pKM-cryIAc. The size of the expected hybridization band was more than 4.2 kb. We detected one to three bands larger 4.2 kb hybridized to the specific cry1Ab or cry1Ac probe in all the PCRconfirmed regenerated plants, but not in the control plants (Fig. 3), indicating the possible integration of the cry1Ab or cry1Ac gene. Among the 65 confirmed R0 plants, 47 (72.3%) showed one hybridization band, suggesting a single insertion site. Thirteen (20%) were detected with two hybridization bands and 5 (7.7%) with three hybridization bands, indicating the presence of two or three insertion sites, respectively. Expression of cry1Ab and cry1Ac The R0 plants confirmed by PCR and Southern blot analysis were subjected to Western blotting analysis
Fig. 2A, B PCR analysis of selected R0 plants of Brassica campestris ssp. parachinensis. A cry1Ab-transformed plants, B cry1Ac-transformed plants. M 1-kb DNA ladder, lanes 1–10 transgenic plants, lane 11 nontransformed plant
Fig. 3 Southern blotting analysis of selected R0 plants of Brassica campestris ssp. parachinensis. M lDNA/HindIII DNA marker, lane 1 binary vector pKM-CryIAb, lane 2 nontransformed plantl, lane 3 cry1Ab-transformed plant, BN-14, lane 4 cry1Ac-transformed plant, BN-18, lane 5 cry1Ab-transformed plant, BN-58, lane 6 cry1Ac-transformed plant, BN-26, lane 7 cry1Ac-transformed plant, BN-1, lane 8 cry1Ab-transformed plant, BN-33, lane 9 cry1Ac-transformed plant, BN-17
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Fig. 4 Western blotting analysis of selected R0 plants of Brassica campestris ssp. parachinensis. M Protein molecular weight marker, lanes 1–3 cry1Ab-transformed plants, BN-3, BN-14 and BN-58, respectively, lanes 4–5 cry1Ac-transformed plants, BN-18 and BN-33, respectively, lane 6 nontransformed plant
using polyclonal B.t. toxin antiserum to detect the presence of the B.t. toxin protein. Proteins extracted from the R0 plants were separated by PAGE gel, and a protein band of about 68 kDa was immunologically detected, thereby positively confirming the presence of the B.t. toxin protein (Fig. 4). The non-transformed plant did not express this 68-kDa protein. From the 65 PCR-and Southern-confirmed R0 plants, 40 (61.5%) were detected as producing the B.t. toxin protein. The remaining transformed plants failed to express the insecticidal protein in spite of the fact that the cry1Ab and cry1Ac genes had been identified in the genomic DNA by PCR and Southern analysis.
Fig. 5 Insect-resistance bioassay of R0 plants of Brassica campestris ssp. parachinensis. Left a cryIAc-transformed plant, BN-14, Right a nontransformed control plant
mortality, leaving behind severely damaged plants (Fig. 5). Among 40 test plants, 10 plants were classified as highly resistance, 23 plants showed moderate resistance and 7 plants were susceptible to larval damage (Table 3).
Discussion Insecticidal activity The R0 plants expressing the B.t. toxin protein were selected for insect bioassay. Insecticidal activity was evaluated by placing larvae of the diamondback moth (Plutella xylostella) on the test plants. We observed that the larvae often bored small holes on the first or second day of feeding on the R0 plants positively identified by Western blot analysis. Two days later, some larvae died and the others ceased feeding and became stunted, depending on the age of the larvae. After 7 days, no surviving larvae were observed on those plants showing high resistance; these plants suffered very little feeding damage in the leaves, stems and flowers (Fig. 5). Some other plants showed moderate resistance where larvae were stunted and quick to pupate. However, the larvae on the non-transformed control plants and susceptible plants continued to feed and grew well to more than 0.7 cm in length and green in appearance after 7 days of feeding. Eventually, these larvae pupated later without
In this study, an efficient Agrobacterium transformation system was established using a 2-day pre-culture of hypocotyl and a 2-day co-cultivation of hypocotyls with Agrobacterium strain MOG301. In general, dicotyledonous plants are easily infected with Agrobacterium. However, hypocotyls from the five varieties of Brassica campestris ssp. parachinensis responded with varying degrees of susceptibility to different Agrobacterium strains. Three Agrobacterium strains, MOG101, MOG301 and EHA105, were tested. MOG301 was found to produce the best results (data not shown). Identification of the integration site by Southern blot analysis of BamHI-digested genomic DNA indicated that most of the R0 plants contained one site and only a few had two or three sites (Fig. 3). These results are consistent with results from other transgenic plants using Agrobacterium-mediated transformation (Cheng et al. 1998).They are in contrast to the general occurrence of multiple sites of integration resulting from direct gene transfer method (Czernilofsky et al. 1986).
Table 3 Insect bioassay of R0 plants of Brassica campestris ssp. parachinensis transformed with the B.t. cry1Ab or cry1Ac gene Gene a
Highly resistant
Moderately resistant
Susceptible
Not determined
Cry1Ab Cry1Ac Total number
2 8 10
10 13 23
2 5 7
18 7 25
a
Highly resistant, No survival of larvae; moderately resistant, larvae stunted; Susceptible, larvae advanced, normal pupae
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In the insect bioassay, the varying degree of resistance among the transgenic plants (Table 3) may obviously result from differences in the expression of the transgene. These differences are consistent with the plant-to-plant variation observed in the expression of other chimeric genes in transgenic plants (Jones et al. 1985). In this study, about 40% of the R0 plants confirmed by PCR and Southern blot analysis could not express the B.t. insecticidal protein. Numerous studies have reported and proposed the mechanism of inability and instability in the expression of transgene (Finnegan and McElroy 1994; Meyer 1995). This gene silencing phenomena is a common problem resulting from gene transfer procedures. It can occur at the transcriptional level by DNA methylation or post-transcriptional level by co-suppression. Factors affecting gene silencing include gene copy numbers, insertion position, host genotype and plant growth conditions (Finnegan and McElroy 1994; Meyer 1995). This study may provide suitable material for further study in the effect or mechanism responsible for the non-expression of cry1Ab and cry1Ac genes in the transformed plants. B.t. crystal proteins have been proven to be very effective in controlling insect damage in crop plants (Hofte and Whiteley 1989). Since the 1980s, insecticidal genes have been transferred to many agricultural crops (Schuler et al. 1998), resulting in highly insect-resistant plants. This study has extended the application of the cry1Ab and cry1Ac genes to Brassica campestris ssp . parachinensis. Acknowledgements This research was supported by a grant from the Biotechnology Research Institute at Hong Kong University of Science & Technology.
References Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1994) Current protocols in molecular biology, vol 2. J Wiley, New York Barfield DG, Pua EC (1991) Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation. Plant Cell Rep 10 : 308–314 Cheng XY, Sardana R, Kaplan H, Altosaar I (1998) Agrobacterium-transformed rice plants expressing synthetic cry1Ab and cry1Ac genes are highly toxic to striped stem borer and yellow stem borer. Proc Natl Acad Sci USA 95 : 2767–2772 Chilton MD, Currier TC, Farrand SK, Bendich AJ, Gordon MP, Nester EW (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci USA 71 : 3672–3676 Czernilofsky AP, Hain R, Baker B, Wirtz U (1986) Studies of structure and functional organization of foreign DNA integrated into the genome of Nicotiana tabacum. DNA 5 : 473–482 De Block M (1988) Genotype-independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens. Theor Appl Genet 76 : 767–774 Finnegan J, McElroy D (1994) Transgenic inactivation: Plants fight back! Bio/technology 12 : 883–888
Hofte H, Whiteley HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53 : 242–255 Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgen Res 2 : 208–218 Jain RK, Chowdhury JB, Sharma DR, Friedt W (1988) Genotypic and media effects on plant regeneration from cotyledon explant cultures of some Brassica species. Plant Cell Tissue Organ Cult 14 : 197–206 Jones JDG, Dunsmuir P, Bedbrook J (1985) High level expression of introduced chimeric genes in regenerated transformed plants. EMBO J 4 : 2411–2418 Jun SI, Kwon SY, Paek KY, Paek KH (1995) Agrobacteriummediated transformation and regeneration of fertile transgenic plants of Chinese cabbage (Brassica campestris ssp. p ekinensis cv ‘Spring flavor’). Plant Cell Rep 14 : 620–625 Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, Dawson J, Desai N, Hill M, Kadwell S, Launis K, Lewis K, Maddox D, McPherson K, Meghji MR, Merlin E, Rhodes R, Warren GW, Wrights M, Evola SV (1993) Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/ technology 11 : 194–200 Metz TD, Dixit R, Earle ED (1995) Agrobacterium tumefaciensmediated transformation of broccoli (Brassica oleracea var ‘italica’) and cabbage (B. oleracea var ‘capitata’). Plant Cell Rep 15 : 287–292 Meyer P (1995) Understanding and controlling transgene expression. Trends Biotechnol 13 : 332–337 Mukhopadhyay A, Arumugam N, Nandakumar PBA, Pradhan AK, Gupta V, Pental D (1992) Agrobacterium-mediated genetic transformation of oilseed Brassica campestris: transformation frequency is strongly influenced by the mode of shoot regeneration. Plant Cell Rep 11 : 506–513 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15 : 473–479 Narashimhulu SB, Chopra VL (1988) Species specific shoot regeneration response of cotyledonary explaints of Brassicas. Plant Cell Rep 7 : 104–106 Perlak FJ, Deaton RW, Armstrong TA, Fuchs RL, Sims SR, Greenplate JT, Fischhoff DA (1990) Insect resistant cotton plants. Bio/Technology 8 : 939–943 Perlak FJ, Fuchs RL, Dean DA, McPherson SL, Fischoff DA (1991) Modification of the coding sequence enhances plant expression of insect control proteins. Proc Natl Acad Sci USA 88 : 3324–3328 Perlak FJ, Stone TB, Muskopf YM, Petersen LJ, Parker GB, McPherson SA, Wyman J, Love S, Reed G, Biever D, Fischoff DA (1993) Genetically improved potatoes: protection from damage by Colorado potato beetle. Plant Mol Biol 22 : 313–321 Radke SE, Andrews BM, Moloney MM, Crouch ML, Kridl JC, Knauf VC (1988) Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor Appl Genet 75 : 685–694 Schuler TH, Poppy GM, Kerry BR, Denholm L (1998) Insectresistant transgenic plants. Trends Biotechnol 16 : 168–175 Swanson EB, Erickson LR (1989) Haploid transformation in Brassica napus using an octopine-producing strain of Agrobacterium tumefaciens. Theor Appl Genet 78 : 831–835 Xiang YB, Wong WKR, Wong RSC (1997) Construction of synthetic genes encoding Bacillus thuringiensis endotoxin for transgenic vegetables. In: Book of Abstracts (Edited by Jeffrey E.D. Dean) 5th Int Congr Plant Mol Biol. 21–27 September Singapore. Abstr no.1314. Supplement to Plant Molecular Biology Reporter, Kluwer Academic Publishers
