Transgenic Broccoli Research Articles

Engineering Brassica Crops to Optimize Delivery of Bioactive Products Postcooking.

Glucosinolates are plant-specialized metabolites that can be hydrolyzed by glycosyl hydrolases, called myrosinases, creating a variety of hydrolysis products that benefit human health. While cruciferous vegetables are a rich source of glucosinolates, they are often cooked before consumption, limiting the conversion of glucosinolates to hydrolysis products due to the denaturation of myrosinases. Here we screen a panel of glycosyl hydrolases for high thermostability and engineer the Brassica crop, broccoli (Brassica oleracea L.), for the improved conversion of glucosinolates to chemopreventive hydrolysis products. Our transgenic broccoli lines enabled glucosinolate hydrolysis to occur at higher cooking temperatures, 20 °C higher than in wild-type broccoli. The process of cooking fundamentally transforms the bioavailability of many health-relevant bioactive compounds in our diet. Our findings demonstrate the promise of leveraging genetic engineering to tailor crops with novel traits that cannot be achieved through conventional breeding and improve the nutritional properties of the plants we consume.

Improving sulforaphane content in transgenic broccoli plants by overexpressing MAM1, FMOGS–OX2, and Myrosinase

Improving sulforaphane content in transgenic broccoli plants by overexpressing MAM1, FMOGS–OX2, and Myrosinase

Ectopic Overexpression of bol-miR171b Increases Chlorophyll Content and Results in Sterility in Broccoli ( Brassica oleracea L var. italica).

MiR171 plays pleiotropic roles in the growth and development of several plant species. However, the mechanism underlying the miR171-mediated regulation of organ development in broccoli remains unknown. In this study, bol-miR171b was characterized and found to be differentially expressed in various broccoli organs. The ectopic overexpression of bol-miR171b in Arabidopsis affected the leaf and silique development of transgenic lines. In particular, the chlorophyll content of leaves from overexpressed bol-miR171b transgenic Arabidopsis was higher than that of the vector controls. The fertility and seed yield of Arabidopsis with overexpressed bol-miR171b were markedly lower than those of the vector controls. Similarly, overexpressed bol-miR171b transgenic broccoli exhibited dark green leaves with high chlorophyll content, and nearly all of the flowers were sterile. These results demonstrated that overexpression of bol-miR171b could increase the chlorophyll content of transgenic plants. Degradome sequencing was conducted to identify the targets of bol-miR171b. Two members of the GRAS gene family, BolSCL6 and BolSCL27, were cleaved by bol-miR171b-3p in broccoli. In addition to the genes targeted by bol-miR171b-3p, adenylylsulfate reductase 3 ( APSR3), which played important roles in plant sulfate assimilation and reduction, was speculated to be cleaved by bol-miR171b-5p, suggesting that the star sequence of bol-miR171b may also have functions in broccoli. Comparative transcriptome analysis further revealed that the genes involved in chloroplast development and sulfate homeostasis should participate in the bol-miR171b -mediated regulatory network. Taken together, these findings provided new insights into the function and regulation of bol-miR171b in broccoli and indicated the potential of bol-miR171b as a small RNA molecule that increased leaf chlorophyll in plants by genetic engineering.

Development of transgenic broccoli with cryIAa gene for resistance against diamondback moth (Plutella xylostella).

Transgenic broccoli (Brassica oleracea L. var. italica) cv. 'Solan Green Head' has been developed by using Agrobacterium tumefaciens strain harboring cryIAa gene for resistance against diamondback moth (Plutella xylostella). By combining the best treatments of 72h pre-culturing and 48h co-cultivation time period, a transformation frequency of 4.92 and 13.83% was obtained from cotyledon and hypocotyl explants, respectively. Supplementing the co-cultivation medium with acetosyringone in a concentration of 100µM enhanced the transformation frequency to 17.92 and 32.11% in cotyledon and hypocotyl explants, respectively. The transgene (cryIAa) integration was confirmed by polymerase chain reaction using gene-specific primers and Southern blot analysis using digoxigenin nonradiolabelled DNA probe. Gene expression in the PCR-positive transgenic events had been confirmed by reverse transcriptase-PCR and quantitative real time-PCR. Insect bioassay proved the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae. To the best of our knowledge, this is the first report of optimization of a highly efficient transformation system and transgenic development in broccoli using cry1Aa gene for insect resistance.

Over-expression of a C3H-type zinc finger gene contributes to salt stress tolerance in transgenic broccoli plants

C3H-type zinc finger proteins play important roles in plant growth, development, and stress responses. A C3H-type zinc finger gene, designated BoC3H, was isolated from broccoli (Brassica oleracea var. italica). The complete coding sequence of BoC3H was 1074 bp in length, encoding 357 amino acids with two CCCH motifs of C-X7-C-X5-C-X3-H and C-X5-C-X4-C-X3-H. The transcripts of BoC3H were profoundly induced by NaCl, and the highest expression level was observed at 18 h after treatment. Four broccoli lines over-expressing the BoC3H gene were obtained, and they exhibited higher germination rates, dry weight and chlorophyll content in response to salt stress as compared to those of wild type plants. Over-expression of BoC3H significantly decreased hydrogen peroxide (H2O2) level, relative electrical conductivity (REC) and malondialdehyde (MDA) contents, but dramatically increased free proline content, catalase, peroxidase and superoxide dismutase enzyme activities, resulting in less cell death in the leaves of transgenic plants. Taken together, our results suggest that BoC3H is likely to contribute to salt stress tolerance by regulating H2O2, REC, free proline, MDA and antioxidant enzyme levels in broccoli.

Over-expression of a WRKY transcription factor gene BoWRKY6 enhances resistance to downy mildew in transgenic broccoli plants

WRKY transcription factors play an important role in plant growth, development and immunity. In our study, a WRKY family member gene designated BoWRKY6 was isolated from broccoli (Brassica oleracea var. italica), and its expression was induced by downy mildew (Hyaloperonospora parasitica). Five transgenic broccoli lines over-expressing BoWRKY6 driven by the CaMV 35S promotor were obtained by Agrobacterium tumefaciens mediated transformation, and they demonstrated significant increased resistance to downy mildew, with resistant levels from low to very high. Real time-qualitative PCR analysis indicated that expressions of both BoWRKY6 and the pathogenesis-related gene 1 (BoPR1) in transgenic plants were obviously higher than those in WT plants after H. parasitica treatment. Lines of BWK14 and BWK31 exhibited very high resistance to downy mildew, and may serve as promising candidate materials for broccoli molecular breeding in the near future.

Changes in Sulforaphane and Selenocysteine Methyltransferase Transcript Levels in Broccoli Treated with Sodium Selenite

Broccoli (Brassica oleracea L. var. italica planck) has been highly valued because of its nutrient content, which has been attributed to both sulforaphane, a sulfur-containing compound, and selenium enrichment ability. The sulforaphane synthesis may be affected by Se/S metabolism because sulfate and selenate share the same initial pathway for uptake in plants. Selenocysteine methyltransferase (SMT) plays a critical role in the Se/S metabolism system. In the present work, we analyzed the SMT expression level and sulforaphane content in transgenic broccoli under the conditions of overexpression and RNAi knockdown of SMT under normal and selenium-stressed conditions. The relative expression value of SMT in the overexpression line is 13.93 % higher than that in the untransformed control. Interestingly, the sulforaphane content of the overexpression line was 14.09 % lower after selenium treatment, while that of the empty vector (pCAMBIA1301) transformed and untransformed plants was 59.04 and 66.56 % lower than that of the non-selenium-treated, respectively. In the SMT-RNAi line, which has a relative expression value for SMT 15.60 % lower than that of the untransformed, we cannot detect sulforaphane after selenium treatment. These results showed that SMT plays a key role in sulforaphane synthesis in a selenium-rich environment. Specifically, overexpression of SMT decreases the negative effect of selenium on sulforaphane synthesis, while knockdown of SMT by RNAi enhances the negative effect.

Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel.

An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05-0.1mgdm(-3) TDZ. TDZ at 0.1mgdm(-3) produced among the highest percentage of shoot regeneration (96.67%) and mean number of shoot formation (6.17). The highest percentage (13.33%) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33%. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.

Expression of An Antisense Brassica oleracea GIGANTEA (BoGI) Gene in Transgenic Broccoli Causes Delayed Flowering, Leaf Senescence, and Post-Harvest Yellowing Retardation

Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop all over the world. However, rapid post-harvest senescence in harvested floral heads reduces its value. Mutation in GIGANTEA (GI) caused delay of flowering and increased tolerance level to H2O2-induced oxidative stress in Arabidopsis. BoGI, a GI orthologue, was isolated and characterized from B. oleracea. BoGI mRNA is expressed throughout development and can be detected in leaves, stem, root, and flowers. Further analysis indicated that the expression of BoGI is modulated by the circadian clock. To investigate the senescence flowering-associated mechanism regulated by BoGI gene and the agricultural application of BoGI in controlling flowering time and floret yellowing for B. oleracea, constructs containing antisense cDNA of BoGI driven by 35S or a flower-specific AP1 promoter were transformed into B. oleracea and the transgenic plants were generated. The flowering time and the senescence of the detached leaves were significantly delayed in transgenic 35S::BoGI antisense plants. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that clear reduction of BoGI expression was observed in these 35S::BoGI antisense plants compared to that in wild-type plants. Furthermore, post-harvest yellow and flower senescence was delayed in AP1::BoGI antisense plants. These findings indicate that BoGI could be involved in regulation of flowering time, leaf, floret, and flower senescence in broccoli.

Homocysteine S-methyl transferase regulates sulforaphane concentration inBrassica oleraceavar.italica

Homocysteine S-methyl transferase (HMT, EC 2.1.1.10) is a ubiquitous enzyme in angiosperms that plays an important role in sulfur nutrition status. We used over-expression and RNA interference (RNAi) to study the regulatory function of BoHMT1 on sulforaphane concentration in broccoli. Agrobacterium-mediated transforrmation was used to produce 63 putatively transgenic broccoli plants; of these, 51 were positive for the gene construct by PCR selection, and 44 were confirmed by GUS staining. Overexpression of BoHMT1 caused a significant increase in the relative expression level of this gene, and five independent transgenic plants had expression levels >4-fold higher than in the control and they also showed an increase in sulforaphane concentration. Similarly, expression of an RNAi construct decreased the relative expression of BoHMT1, and four independent transgenic lines had expression levels >4-fold lower than in the control, and also showed a reduction in sulforaphane concentration. It is concluded that the BoHMT1 gene regulates sulforaphane concentration in broccoli and that there is a positive relationship between the expression level of the gene and the sulforaphane content.

Compositional and proteomic analyses of genetically modified broccoli (Brassica oleracea var. italica) harboring an agrobacterial gene.

Previously, we showed improved shelf life for agrobacterial isopentenyltransferase (ipt) transgenic broccoli (Brassica oleracea var. italica), with yield comparable to commercial varieties, because of the protection mechanism offered by molecular chaperones and stress-related proteins. Here, we used proximate analysis to examine macronutrients, chemical and mineral constituents as well as anti-nutrient and protein changes of ipt-transgenic broccoli and corresponding controls. We also preliminarily assessed safety in mice. Most aspects were comparable between ipt-transgenic broccoli and controls, except for a significant increase in carbohydrate level and a decrease in magnesium content in ipt-transgenic lines 101, 102 and 103, as compared with non-transgenic controls. In addition, the anti-nutrient glucosinolate content was increased and crude fat content decreased in inbred control 104 and transgenic lines as compared with the parental control, “Green King”. Gel-based proteomics detected more than 50 protein spots specifically found in ipt-transgenic broccoli at harvest and after cooking; one-third of these proteins showed homology to potential allergens that also play an important role in plant defense against stresses and senescence. Mice fed levels of ipt-transgenic broccoli mimicking the 120 g/day of broccoli eaten by a 60-kg human adult showed normal growth and immune function. In conclusion, the compositional and proteomic changes attributed to the transgenic ipt gene did not affect the growth and immune response of mice under the feeding regimes examined.

Greenhouse evaluation on performance of transgenic broccoli and genetic diversity analysis of the transgenic and non-transgenic plants using Inter Simple Sequence Repeat (ISSR) markers

Background: Broccoli, Brassica oleracea subsp. italica is one of the many valuable Brassica species which is still less cultured under in vitro condition. Heat tolerant transgenic and non-transgenic broccoli cv. Green Marvel plantlets with well-developed root system obtained through in vitro culture were transferred into disposable plastic pots containing sterilized potting mixture consisting of (peatgroTM) + coconut dust (2:1) and maintained in a growth chamber. Results: After one month, the hardened plantlets were transferred and maintained in a transgenic greenhouse. After four months of acclimatization in the transgenic greenhouse, the efficacy of HSP101 gene in increasing the heat tolerance of the transgenic broccoli was evaluated. Results showed that the transgenic plants could survive and performed normally, producing flower heads even at the highest tested temperature of 34oC. Seven transgenic broccoli lines with different gene copy number of the AtHSP101 gene as well as the control plant were assessed for genetic diversity using inter simple sequence repeat (ISSR) markers. Conclusions: ISSR results showed polymorphism and phylogenetic relationship between the transgenic and non-transgenic (control) Brassica oleracea cv. Green Marvel.

Proteomics and transcriptomics of broccoli subjected to exogenously supplied and transgenic senescence-induced cytokinin for amelioration of postharvest yellowing

Previously, we investigated transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and mimics the action of exogenous supplied CK in delaying postharvest senescence of broccoli. Here, we used proteomics and transcriptomics to compare the mechanisms of ipt-transgenic and N(6)-benzylaminopurine (BA) CK treatment of broccoli during postharvest storage. The 2 treatments conferred common and distinct mechanisms. BA treatment decreased the quantity of proteins involved in energy and carbohydrate metabolism and amino acid metabolism, and ipt-transgenic treatment increased that of stress-related proteins and molecular chaperones and slightly affected levels of carbohydrate metabolism proteins. Both treatments regulated genes involved in CK signaling, sugar transport, energy and carbohydrate metabolism, amino acid metabolism and lipid metabolism, although ipt-transgenic treatment to a lesser extent. BA treatment induced genes encoding molecular chaperones, whereas ipt-transgenic treatment induced stress-related genes for cellular protection during storage. Both BA and ipt-transgenic treatments acted antagonistically on ethylene functions. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. Transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and N(6)-benzylaminopurine (BA) CK treated broccoli both showed retardation of postharvest senescence during storage. The mechanisms underlying the two treatments were compared. The combination of proteomic and transcriptomic evidences revealed that the 2 treatments conferred common and distinct mechanisms in delaying senescence of broccoli florets. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Proteomic analysis of stress-related proteins in transgenic broccoli harboring a gene for cytokinin production during postharvest senescence

Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli ( Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase ( ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography–electrospray ionization–mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis–trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days’ post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence.

Growth, yield and shelf-life of isopentenyltransferase (ipt)-gene transformed broccoli

Loss of chlorophyll leading to floret yellowing limits the post-harvest lifespan of broccoli (Brassica oleracea L. var. italica Plenck). Cytokinins are known to delay floral yellowing of plants. A transgene construct pSG766A, which results in the expression of isopentenyltransferase (ipt), the key enzyme for cytokinin synthesis, has been developed in broccoli. Expression of the ipt transgene is triggered by the senescence-associated gene promoter (SAG-13). Three selfed T5 lines of ipt transformed broccoli (lines 101, 102 and 103) have been obtained through selection for single copy insertion, acceptable horticultural traits and transgene ipt activity. These three transgenic inbred lines were evaluated in the field during 2004-2007 to determine their growth, yield and shelf-life after harvest, relative to a non-transgenic inbred line (104) and the parental variety Green King. For most of the vegetative growth parameters measured, year-to-year variability exceeded line-to-line variability. Inbreeding had little impact on the appearance or yield potential of the broccoli lines. Head yields of the transgenic inbred lines 102 and 103 were comparable to the parental variety Green King, but were significantly higher than the non-transgenic inbred line 104, as lines 102 and 103 produced more plants with heavier flower heads. Cytokinin content in the form of isopentenyladenosine was relatively higher in the transgenic lines than in the two non-transgenic controls. When flower heads were stored at 25 ± 2°C, the period required to cause 50% floret yellowing was 7.5 and 8.5 d for the transgenic lines 102 and 103, respectively, compared with 5.6 d for the non-transgenic line 104, and 5.1 d for the parental variety Green King. This study showed that the ipt-transformed inbred lines of broccoli combined acceptable appearance and yields with enhanced shelf-life.Key words: Brassica oleracea L. var. italica Plenck, transgenic broccoli, isopentenyltransferase gene, genetic characterization, shelf-life

Suppressing expression of a soluble acid invertase (BoINV2) in broccoli (Brassica oleracea) delays postharvest floret senescence and downregulates cysteine protease (BoCP5) transcription

We report on the production and selection of transgenic Brassica oleracea var. Italica lines with a downregulated soluble acid invertase (BoINV2). Explants of broccoli (cv. Triathlon) were transformed with an antisense construct of BoINV2 under the control of an Asparagus officinalis‐derived harvest‐induced promoter using Agrobacterium tumefaciens‐mediated transformation. BoINV2 is upregulated in wild‐type broccoli floret tissue after harvest. Transgenic broccoli lines showed reduced BoINV2 mRNA accumulation immediately after harvest compared with wild‐type. Downregulation of BoINV2 had no significant impact on the expression of a second broccoli acid invertase gene (BoINV1), but plants with downregulated BoINV2 also had lower expression of a senescence‐associated cysteine protease (BoCP5) compared with wild‐type. The total soluble sugar levels in floret tissue of antisense BoINV2 lines were greater than wild‐type tissue after harvest (up to 1.5 times higher). Soluble protein content of wild‐type tissue decreased from 48 h after harvest with an increase in protease activity. In comparison, two antisense BoINV2 lines retained at‐harvest levels of soluble protein until 72 and 96 h after harvest and had lower postharvest endoprotease activity compared with wild‐type. Antisense BoINV2 lines also had a slower rate of floret sepal chlorosis after harvest compared with wild‐type.

BO-ACO2 遺伝子組み込みがブロッコリーの内生ホルモンに及ぼす影響

BO-ACO2 遺伝子組み込みがブロッコリーの内生ホルモンに及ぼす影響

The Production of Marker-Free Genetically Engineered Broccoli with Sense and Antisense ACC synthase 1 and ACC oxidases 1 and 2 to Extend Shelf-Life

The production of transgenic broccoli (Brassica oleracea) with increased shelf-life using an Agrobacterium rhizogenes-mediated co-transformation protocol is reported. An Agrobacterium rhizogenes Ri vector, pRi1855:GFP was constructed to allow expression of the green fluorescent protein to identify insertion of Ri TL-DNA into plant cells. The Brassica oleracea ACC synthase 1 and ACC oxidase 1 and 2 cDNAs in sense and antisense orientations were co-transformed into GDDH33, a doubled haploid calabrese-broccoli cultivar. Transformation efficiency was 3.26%, producing 150 transgenic root lines, of which 18 were regenerated into mature plants. The floral buds from T0 broccoli heads were assayed for post-harvest production of ethylene and chlorophyll levels. Buds from T0 lines transformed with ACC oxidase 1 and 2 constructs produced significantly less post-harvest ethylene at 20 °C than the untransformed plants and chlorophyll loss was significantly reduced over a 96 h post-harvest period. The T0 plants transformed with sense and antisense ACC synthase 1 had a significantly reduced 24 h post-harvest ethylene peak and delayed chlorophyll loss. A positive correlation between post-harvest bud ethylene production and chlorophyll loss was described by a regression. This demonstrates that the shelf-life of a very perishable vegetable may be increased up to 2 days at 20 °C by reducing post-harvest ethylene production.

Evaluation of a Chemically Inducible Promoter for Developing a Within-Plant Refuge for Resistance Management

Chemically inducible production of Bacillus thuringiensis (Bt) toxins in transgenic plants may provide considerable benefits in preventing or delaying the evolution of insect resistance to Bt crops by creating within-plant temporal refuges. We examined the effect of inducible cry1Ab expression on survival of different genotypes (RR, RS, and SS) of diamondback moth, Plutella xylostella (L.), in transgenic broccoli, Brassica oleracea L., plants transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-s-methyl [=benzo (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester] (ASM) resulted in high levels of Bt toxin, and detached leaves from fully induced plants caused 100% mortality to all instars of P. xylostella SS and RS genotypes. When plants infested with larvae were treated with ASM, only a few larvae that were nearing completion of their development were able to survive the induction process. Signal transduction from ASM-treated leaves to new plant tissue also was evaluated using a larval assay. New foliage that emerged after plants were induced remained toxic to ≥80% of RS larvae up to the fourth new leaf. In whole plant tests, however, induced plants remained protected from larval damage for ≥3 wk. Uninduced PR-1a/cry1Ab plants seemed to produce low levels of Bt that were undetected by an enzyme-linked immunosorbent assay but that resulted in significant fitness costs for susceptible insects. The suitability of PR-1a/cry1Ab broccoli plants for insect resistance management and the requirements of an appropriate inducible promoter are discussed.

Evaluation of a Chemically Inducible Promoter for Developing a Within-Plant Refuge for Resistance Management

Chemically inducible production of Bacillus thuringiensis (Bt) toxins in transgenic plants may provide considerable benefits in preventing or delaying the evolution of insect resistance to Bt crops by creating within-plant temporal refuges. We examined the effect of inducible cry1Ab expression on survival of different genotypes (RR, RS, and SS) of diamondback moth, Plutella xylostella (L.), in transgenic broccoli, Brassica oleracea L., plants transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-s-methyl [= benzo (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester] (ASM) resulted in high levels of Bt toxin, and detached leaves from fully induced plants caused 100% mortality to all instars of P. xylostella SS and RS genotypes. When plants infested with larvae were treated with ASM, only a few larvae that were nearing completion of their development were able to survive the induction process. Signal transduction from ASM-treated leaves to new plant tissue also was evaluated using a larval assay. New foliage that emerged after plants were induced remained toxic to > or = 80% of RS larvae up to the fourth new leaf. In whole plant tests, however, induced plants remained protected from larval damage for > or = 3 wk. Uninduced PR-1a/cry1Ab plants seemed to produce low levels of Bt that were undetected by an enzyme-linked immunosorbent assay but that resulted in significant fitness costs for susceptible insects. The suitability of PR-1a/cry1Ab broccoli plants for insect resistance management and the requirements of an appropriate inducible promoter are discussed.