Transgenic broccoli with high levels of Bacillus thuringiensis Cry1C protein control diamondback moth larvae resistant to Cry1A or Cry1C

A synthetic Bacillus thuringiensis (Bt) cry1C gene was introduced into broccoli (Brassica oleracea ssp. italica) by Agrobacterium-mediated transformation. Twenty-one Cry1C transgenic plants were regenerated from 400 hypocotyl and petiole explants. Variable amounts of stable steady- state cry1C mRNA accumulated in different transgenic plants. Cry1C protein (up to 0.4% of total soluble protein) was produced in correlation with the cry1C mRNA levels. Leaf section and whole-plant bioassays were done using diamondback moth (DBM) larvae from lines susceptible to Bt or resistant to Cry1A or Cry1C proteins (Cry1AR or Cry1CR, respectively). Plants with high levels of Cry1C protein caused rapid and complete mortality of all three types of DBM larvae with no defoliation. Plants with lower levels of Cry1C protein showed an increasing differential between control of susceptible of Cry1AR DBM. This study demonstrated that high production of Cry1C protein can protect transgenic broccoli not only from susceptible or Cry1AR DBM larvae but also from DBM selected for moderate levels of resistance of Cry1C. The Cry1C- transgenic broccoli were also resistant to two other lepidopteran pests of crucifers (cabbage looper and imported cabbage worm). These plants will be useful in studies of resistance management strategies involving multiple transgenes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Insect-resistant chrysanthemum calluses by introduction of aBacillus thuringiensis crystal protein gene

A 3-end truncated crystal protein gene, derived fromBacillus thuringiensis (Bt) subsp.aizawai 7.21, encoding the toxic fragment of the insecticidal proteincryIA(b), was constructed. The gene was inserted into a transformation vector, also carrying the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (gus) gene, and introduced in the oncogenicAgrobacterium tumerfaciens strain A281, harbouring the Ti-plasmid pTiBO542. The recombinantAgrobacterium strain was used to transform leaf explants of chrysanthemum (Dendranthema grandiflora) cultivar Parliament. The resulting tumours were kanamycin-resistant, exhibited -glucuronidase activity and produced agropine and mannopine. In most tumours, all simultaneously transferred genes were expressed, owing to selection for the presence of both T-DNAs, but no correlation was found between the level of expression of the various genes. A bioassay was developed, in which larvae were fed with tumorous chrysanthemum tissue, in order to detect the effect of the transferred toxin gene on larval development. Using this bioassay with second instar larvae ofHeliothis virescens (tobacco budworm), 17 tumour lines were tested. Several of these lines proved to be strongly inhibitory to larval growth. These results indicate thatBt-based insect resistance might be used as a tool in reducing the amount of pesticides used in chrysanthemum culture.     

Recovery and evaluation of soybean plants transgenic for aBacillus thuringiensis var.Kurstaki insecticidal gene

Summary Lepidopteran insects are major defoliating pests of soybean in the southeastern United States. Soybean plants transgenic for a nativecryIA(b) gene fromBacillus thuringiensis var.kurstaki HD-1 were obtained. Embryogenic cultures were induced by plating cotyledons on a Murashige and Skoog-based medium supplemented with 40 mg/liter of 2,4-dichlorophenoxyacetic acid (2,4-D). The embryogenic cultures were maintained in liquid medium containing 5 mg/liter 2,4-D. These cultures were subjected to microprojectile bombardment, followed by selection on 50 mg/liter hygromycin. Resistant embryogenic cell lines were transferred to growth regulator-free medium to permit recovery of mature somatic embryos. After a desiccation period, the somatic embryos were returned to growth regulator-free medium for conversion into plants. Southern hybridization analysis verified transformation. Feeding assays of T₁ plants from one cell line deterred feeding, development, and survival of velvetbean caterpillar at a level comparable to that of GatIR81-296, a soybean breeding line with a high level of insect resistance. Reduced feeding on T₁ plants correlated with the presence of the transgene.     

Gene expression and insect resistance in transgenic broccoli containing a Bacillus thuringiensis cry1Ab gene with the chemically inducible PR-1a promoter

We produced 49 broccoli plants (Brassica oleracea L. ssp. italica) containing a Bacillus thuringiensis cry1Ab gene under control of the chemically inducible PR-1a promoter from tobacco. Most of them showed substantial or complete control of neonate diamondback moth larvae, regardless of whether the transgene was induced or not. Ten plants were selected for detailed study via northern and western analysis and insect bioassays. They expressed the cry1Ab gene and gave complete insect control when treated with the chemical inducers INA (2,6-dichloroiso-nicotinic acid) or BTH (1,2,3-benzothiadiazole-7-carbothioic acid S-methyl ester); however, leaves treated with water alone were also partially or completely protected from insect damage. Transgenic progeny plants showed greater inducibility than primary transformants at the molecular level. Two progeny lines produced cry1Ab mRNA and Cry1Ab protein and gave insect control only after induction, both when detached leaves and intact plants were tested. The relevance of these results to resistance management strategies is discussed.     

Bacillus thuringiensis protein production, signal transduction, and insect control in chemically inducible PR-1a/cry1Ab broccoli plants

In an effort to develop a chemically inducible system for insect management, we studied production of Cry1Ab Bacillus thuringiensis (Bt) protein and control of the diamondback moth (DBM), Plutella xylostella L., in inducer-treated and untreated tissues of a broccoli line transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-S-methyl (= 1,2,3 benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester) (ASM) triggered expression of the cry1Ab gene and produced a high level of Cry1Ab protein within 2–3 days. Cry1Ab protein persisted in leaves for at least 8 weeks, providing prolonged protection from P. xylostella attack. Signals generated in inducer-treated leaves were transferred to untreated newly emerged leaves or heads, as seen by production of Cry1Ab protein and/or protection from insect damage in these plant parts. Signal transduction proceeded in an attenuated manner up to the sixth newly emerged leaf. No Cry1Ab protein was detectable by ELISA in uninduced young leaves, but small amounts of the protein were present in uninduced leaves older than 3 weeks and caused some insect mortality. Such basal expression of Bt genes without induction may favor the evolution of resistant insect populations and therefore limits the application of the PR-1a/cry1Ab system for insect management. However, the rapid production and steady maintenance of a high level of transgenic protein upon induction, the signal transduction observed, and the fact that the chemical inducer can be used in field conditions make the PR-1a promoter attractive for chemical regulation of other agriculturally or pharmaceutically important genes for which low expression in the absence of induction is not a concern.     

Behavioral and physiological responses of susceptible and resistant diamondback moth larvae to Bacillus thuringiensis

To determine whether field-selected resistance of diamondback moth (Plutella xylostella L.) (Lepidoptera: Plutellidae) to Bacillus thuringiensis is based on behavioral or physiological adaptation, we measured mortality, consumption, and movement of larvae from a susceptible and a resistant colony when placed on untreated and B. thuringiensis treated cabbage. Colonies did not differ in mortality, consumption, or movement on untreated cabbage. However, for a given amount of consumption of treated cabbage, resistant larvae had lower mortality than susceptible larvae, demonstrating that resistance had a physiological basis. The movement patterns could not account for the differences between colonies in survival. Resistant larvae did not avoid B. thuringiensis more than did susceptible larvae. Thus, we found no evidence for behavioral resistance.     

Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

A sporulating culture ofBacillus thuringiensis subsp.kenyae strain HD549 is toxic to larvae of lepidopteran insect species such asSpodoptera litura, Helicoverpa armigera andPhthorimaea operculella, and a dipteran insect,Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed inE. coli. The recombinant protein produced 92% mortality in first-instar larvae ofSpodoptera litura and 86% inhibition of adult emergence inPhthorimaea operculella, but showed very low toxicity againstHelicoverpa armigera, and lower mortality against third-instar larvae of dipteran insectsCulex fatigans, Anopheles stephensi andAedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene fromBacillus thuringiensis var.kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.     

Application of synthetic sex pheromone for management of diamondback moth, Plutella xylostella, in cabbage

Over a 2-year period field trials were conducted to assess the potential to disrupt mating ofPlutella xylostella (L.) using a commercial rope formulation of a 70:30 mixture of (Z)-11-hexadecenal and (Z)-11-hexadecenyl acetate, two components of the sex pheromone of the female. Screened field cages were placed into blocks of cabbage which were either treated with the pheromone or left untreated. Different densities of P. xylostella pupae were placed into each cage and then larval and pupal counts were made of the subsequent generation. In addition, sentinel females at mating stations were placed in each cage to assess the influence of the pheromone on the ability of males to locate and mate with females. Likewise, we used pheromone traps to assess whether the pheromone treatment influenced the ability of males to locate a pheromone source. In both years larval and pupal populations, produced as a result of the original inoculation, did not differ between pheromone-treated and untreated fields. The effect of pheromone treatment on larval and pupal numbers did not change with changes in inoculated P. xylostella density, however, the density of P. xylostella released caused significant differences in the density of the subsequent generation. No significant differences were detected between the number of sentinel female adult P. xylostella that successfully mated in pheromone-treated fields compared with untreated fields. Significant differences in the numbers of male P. xylostella caught in pheromone-baited traps occurred between pheromone-treated and untreated fields in the first trial of 1993, and in the first trial in 1994 but not in the second trial. Such differences are often thought of as indications of mating disruption, although our other data presented in this study and reports from other studies indicate this is not always the case. Previous studies on mating disruption of P. xylostella in larger scale field tests have been performed but the results have been variable and often ambiguous. Overall, our results indicate that mating disruption of P. xylostella with the present technology does not appear to work even under the very controlled situations which we utilized to eliminate insect movement between plots.     

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco

Insecticidal transgenic tobacco plants containing a truncated Bacillus thuringiensis cryIA(b) crystal protein (ICP) gene expressed from the CaMV 35S promoter were analyzed for ICP gene expression under field and greenhouse conditions over the course of a growing season. We present new information on temporal and tissue-specific expression of a CaMV 35S/cryIA(b) gene. Levels of cryIA(b) protein and mRNA were compared in both homozygous and hemizygous lines throughout plant development. Levels of ICP mRNA and protein increased during plant development with a pronounced rise in expression at the time of flowering. Homozygous ICP lines produced higher levels of ICP than the corresponding hemizygous lines. ELISA analysis of different tissues in the tobacco plant showed ICP gene expression in most tissues with a predominance of ICP in older tissue. All transgenic ICP tobacco lines which were studied in the field and greenhouse contained 400 ng to 1 g ICP per gram fresh weight in leaves from the mid-section of the plant at flowering. The amounts of ICP produced by field lines were directly comparable to levels observed in greenhouse-grown plants.     

Transfer of an insecticidal protein gene ofBacillus thuringiensis into plant-colonizingAzospirillum

A fusion plasmid, pRKC, was constructed, using pACYC184, RSF1010 and a kanamycin-resistance cartridge from pUC4K, to convey thecryIA(a) gene intoAzospirillum spp. With the pRKC plasmid, the number of putative transconjugants obtained inA. lipoferum was about 300-fold higher than inA. brasilense. Conjugation frequency and plasmid stability inA. lipoferum were less for pBTF8, which carries thecryIA(a) gene in the correct orientation for a constitutive promoter, than for pBTF9, which carries the gene in the opposite orientation. Expression of thecryIA(a) gene was not apparent in SDS-PAGE analysis ofA. lipoferum transconjugants harbouring pBTF8. However,Escherichia coli transformants with the pBTF8 rescued fromA. lipoferum transconjugants produced an approximately 135 kDa Cry protein, indicating that thecry gene is intact in the transconjugants.V. Udayasuriyan was and A. Nakamura, H. Masaki and T. Uozumi are with the Department of Biotechnology, Faculty of Agriculture, The University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo 113, Japan; V. Udayasuriyan is now with the Department of Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultral University, Coimbatore-641 003, India.     

Characterization and comparison of midgut proteases of Bacillus thuringiensis susceptible and resistant diamondback moth (Plutellidae: Lepidoptera)

The midgut proteases of the Bacillus thuringiensis resistant and susceptible populations of the diamondback moth, Plutella xylostella L. were characterized by using protease specific substrates and inhibitors. The midgut contained trypsin-like proteases of molecular weights of 97, 32, 29.5, 27.5, and 25 kDa. Of these five proteases, 29.5 kDa trypsin-like protease was the most predominant in activation of protoxins of Cry1Aa and Cry1Ab. The activation of Cry1Ab protoxin by midgut protease was fast (T(1/2) of 23-24 min) even at a protoxin:protease ratio of 250:1. The protoxin activation appeared to be multi-step process, and at least seven intermediates were observed before formation of a stable toxin of about 57.4 kDa from protoxin of about 133 kDa. Activation of Cry1Aa was faster than that of Cry1Ab on incubation of protoxins with midgut proteases and bovine trypsin. The protoxin and toxin forms of Cry proteins did not differ in toxicity towards larvae of P. xylostella. The differences in susceptibility of two populations to B. thuringiensis Cry1Ab were not due to midgut proteolytic activity. Further, the proteolytic patterns of Cry1A protoxins were similar in the resistant as well as susceptible populations of P. xylostella.     

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369–375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.     

The insecticidal crystal protein Cry2Ab10 from Bacillus thuringiensis: cloning, expression, and structure simulation

The cry2Ab-type gene was cloned from Bacillus thuringiensis and designated as cry2Ab10. The recombinant Cry2Ab10 protein expressed in E. coli cells shows high toxicity against Plutella xylostella. The protein structure was constructed by homology modeling, and the receptor-binding sites were predicted by a molecular docking method.     

Implications of transgenic, insecticidal plants for insect and plant biodiversity

Genetically modified plants are widely grown predominantly in North America and to a lesser extent in Australia, Argentina and China but their regions of production are expected to spread soon beyond these limited areas also reaching Europe where great controversy over the application of gene technology in agriculture persists. Currently, several cultivars of eight major crop plants are commercially available including canola, corn, cotton, potato, soybean, sugar beet, tobacco and tomato, but many more plants with new and combined multiple traits are close to registration. While currently agronomic traits (herbicide resistance, insect resistance) dominate, traits conferring “quality” traits (altered oil compositions, protein and starch contents) will begin to dominate within the next years. However, economically the most promising future lies in the development and marketing of crop plants expressing pharmaceutical or “nutraceuticals” (functional foods), and plants that express a number of different genes. From this it is clear that future agricultural and, ultimately, also natural ecosystems will be challenged by the large-scale introduction of entirely novel genes and gene products in new combinations at high frequencies all of which will have unknown impacts on their associated complex of non-target organisms, i.e. all organisms that are not targeted by the insecticidal protein. In times of severe global decline of biodiversity, pro-active precaution is necessary and careful consideration of the likely expected effects of transgenic plants on biodiversity of plants and insects is mandatory.In this paper possible implications of non-target effects for insect and plant biodiversity are discussed and a case example of such non-target effects is presented. In a multiple year research project, tritrophic and bitrophic effects of transgenic corn, expressing the gene from Bacillus thuringiensis (Bt-corn) that codes for the high expression of an insecticidal toxin (Cry1Ab), on the natural enemy species, Chrysoperla carnea (the green lacewing), was investigated. In these laboratory trials, we found prey-mediated effects of transgenic Bt-corn causing significantly higher mortality of C. carnea larvae. In further laboratory trials, we confirmed that the route of exposure (fed directly or via a herbivorous prey) and the origin of the Bt (from transgenic plants or incorporated into artificial diet) strongly influenced the degree of mortality. In choice feeding trials where C. carnea could choose between Spodoptera littoralis fed transgenic Bt-corn and S. littoralis fed non-transgenic corn, larger instars showed a significant preference for S. littoralis fed non-transgenic corn while this was not the case when the choice was between Bt- and isogenic corn fed aphids. Field implications of these findings could be multifold but will be difficult to assess because they interfere in very intricate ways with complex ecosystem processes that we still know only very little about. The future challenge in pest management will be to explore how transgenic plants can be incorporated as safe and effective components of IPM systems and what gene technology can contribute to the needs of a modern sustainable agriculture that avoids or reduces adverse impacts on biodiversity? For mainly economically motivated resistance management purposes, constitutive high expression of Bt-toxins in transgenic plants is promoted seeking to kill almost 100% of all susceptible (and if possible heterozygote resistant) target pest insects. However, for pest management this is usually not necessary. Control at or below an established economic injury level is sufficient for most pests and cropping systems. It is proposed that partially or moderately resistant plants expressing quantitative rather than single gene traits and affecting the target pest sub-lethally may provide a more meaningful contribution of agricultural biotechnology to modern sustainable agriculture. Some examples of such plants produced through conventional breeding are presented. Non-target effects may be less severe allowing for better incorporation of these plants into IPM or biological control programs using multiple control strategies, thereby, also reducing selection pressure for pest resistance development.     

Tackling diamondback moth Plutella xylostella (L.) resistance: a review on the current research on vegetable integrated pest management in Zimbabwe

The diamondback moth (DBM), Plutella xylostella (L.) is a destructive pest of brassicas globally. Control of the pest is dominated by insecticides. Studies have shown that in some African countries, there is a great reliance on broad spectrum insecticides such as pyrethroids, organophosphates and carbamates, that are applied weekly or twice per week. Use of unregistered insecticides has also been reported. The quality of insecticide application has also been reported to be poor or ineffective. It is therefore not surprising that DBM is fast developing resistance to the major insecticides used against it. Adopting an integrated pest management strategy may be a good arsenal to use against the pest.     

Persistence and impact on microorganisms of Bacillus thuringiensis proteins in some Zimbabwean soils

The persistence of the Bacillus thuringiensis subsp. kurstaki (Btk) toxin (Cry1Ab protein) from Bt maize (MON810, Yieldgard®) residues incorporated in a vertisol (739 g clay kg^?1) was investigated. The maize residues were incubated in the soil for 4 weeks, and activity of the toxin in the residues was bioassayed using larvae of the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae). Corrected mortality of P. xylostella in the bioassays decreased from 76% to 30% in less than a week of incubation in the soil. In addition to the above observations, the effects of Btk, Bt subsp. israelensis (Bti), and Bt subsp. tenebrionis (Btt) proteins on the soil microbiota were examined using a vertisol, an alfisol, and an oxisol. The pre-incubated soils (7 days after moisture adjustment) were treated with crystal proteins of Btk, Bti, and Btt and incubated for further a 7-day period. Microbial biomass carbon (MBC) and counts of culturable bacteria and fungi were determined. The proteins did not show effects on MBC or bacterial and fungal counts, possibly as a result of adsorption of the proteins on soil particles, which could have rendered the proteins inaccessible for microbial utilization. Microbial biomass carbon and counts arranged in decreasing order were vertisol>oxisol>alfisol, similar to the amounts of organic C and clay in the soils. However, bacteria and fungi counts were higher in the vertisol than in the alfisol and the oxisol soils. Our observations suggest that larvicidal proteins produced by different subspecies of Bt and Bt maize could persist in tropical soils as a result of adsorption on soil clays but that there were no observable effect on the soil microbiota.     

Waxes enhance Plutella xylostella oviposition in response to sinigrin and cabbage homogenates

Oviposition by the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), on substrates treated with host stimuli (cabbage homogenate or sinigrin) and/or waxes (paraffin or a mixture of 10 single chain n-alkanes) was quantified using continuous observations and endpoint bioassays. Paraffin or an n-alkane mixture applied over cabbage homogenate or sinigrin caused an increase in oviposition compared to that on any single stimulus in choice tests. Sinigrin alone at 10^–5 M to 10^–2 M is an ovipositional stimulant; addition of alkane over sinigrin made all sinigrin concentrations (10^–6 M to 10^–2 M) significantly more stimulatory than controls. Waxes alone do not stimulate oviposition. In choice tests, insect movement between sinigrin/alkane treatment combinations was random, however, once encountered, visit duration was significantly longer on sinigrin with alkane than on sites treated with either stimulus alone. Given the ubiquity of waxes on plant surfaces and the interaction between waxes and host-specific chemical stimuli, waxes should be included when considering factors that significantly influence herbivore host acceptance.     

Fitness costs of resistance to Bacillus thuringiensis in the Indianmeal moth, Plodia interpunctella

Genetic changes in insects that result in insecticide resistance can also affect their fitness. Here, we report measurements of development time and survival of the Indianmeal moth, Plodia interpunctella (Hübner), to compare the relative fitness of Bacillus thuringiensis (Bt)-susceptible and -resistant colonies. Measurements of larval development time and survival indicated that a fitness cost was associated with resistance to Bt in some Bt-resistant colonies but not others. Comparisons of geographically different populations revealed inherent differences in development time and survival. In most cases, Bt-resistant moths suffered no disadvantage when feeding on a Bt-treated diet. In many cases, the development of Bt-resistant moths on Bt-treated diet was slower than the unselected moths on untreated diet, but it is unclear whether these differences would affect the successful mating of susceptible and resistant moths.     

Effects of individual Bacillus thuringiensis insecticidal crystal proteins on adult Heliothis virescens (F.) and Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae)

Bacillus thuringiensis (Bt) is a grampositive, spore forming bacterium, which is principally distinguishedfrom other bacilli by the production of large, insecticidal,protein crystals (Insecticidal Crystal Proteins, or ICPs). Theseproteins are usually thought to act only on the actively feedinglarvae of susceptible species by a mechanism which involvesconsumption and proteolytic processing of the protein followed bybinding to, and lysis of, midgut epithelial cells. However, few authorshave reported Bt toxicity to adult insects. In the followingpaper, we expand on previous reports of toxicity to adult insects andpresent data which demonstrate that: (1) proteolytically activated ICPssignificantly reduce the lifespans of adult Heliothis virescensand Spodoptera exigua at concentrations of 500 g/ml, butnot 167 or 25 g/ml, (2) individual activated ICPs are differentiallytoxic to adult H. virescens and S. exigua, and (3)adult S. exigua are sensitive to Cry1C protoxin at aconcentration of 1 mg/ml.Deceased     

Resistance to Alternaria brassicicola in transgenic broccoli expressing a Trichoderma harzianum endochitinase gene

Transgenic broccoli plants expressing a Trichoderma harzianum endochitinase gene were obtained by Agrobacterium tumefaciens-mediated transformation. PCR and Southern blot analysis confirmed the presence of the gene in plants initially selected via resistance to kanamycin. Primary transformants (T₀) and selfed progeny (T₁) were examined for expression of the endochitinase gene using a fluorometric assay and for their resistance to the fungal pathogens Alternaria brassicicola and Sclerotinia sclerotiorum. All transgenic plants with elevated endochitinase activity had the expected 42 kDa endochitinase band in western blot analysis, whereas no such band was detected in the non-transgenic control. Leaves of most mature T₀ plants had 14–37 times higher endochitinase activity than controls; mature T₁ plants had higher endochitinase activity (100–200 times that in controls), in part because of lower control values. T₀ plantlets in vitro or young plants in soil had higher absolute and relative endochitinase activity. When detached leaves of T₀ plants were inoculated with A. brassicicola, lesion size showed a significant negative correlation with endochitinase levels. After inoculation of two-month old T₀ plants with A. brassicicola, all 15 transgenic lines tested showed significantly less severe disease symptoms than controls. In contrast, lesion size on petioles of T₀ and T₁ plants inoculated with S. sclerotiorum was not statistically different from controls.