Map-based cloning of a protein kinase gene conferring disease resistance in tomato.

The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.

Field tests on managing resistance to Bt-engineered plants.

Several important crops have been engineered to express toxins of Bacillus thuringiensis (Bt) for insect control. In 1999, US farmers planted nearly 8 million hectares (nearly 20 million acres) of transgenic Bt crops approved by the EPA. Bt-transgenic plants can greatly reduce the use of broader spectrum insecticides, but insect resistance may hinder this technology. Present resistance management strategies rely on a "refuge" composed of non-Bt plants to conserve susceptible alleles. We have used Bt-transgenic broccoli plants and the diamondback moth as a model system to examine resistance management strategies. The higher number of larvae on refuge plants in our field tests indicate that a "separate refuge" will be more effective at conserving susceptible larvae than a "mixed refuge" and would thereby reduce the number of homozygous resistant (RR) offspring. Our field tests also examined the strategy of spraying the refuge to prevent economic loss to the crop while maintaining susceptible alleles in the population. Results indicate that great care must be taken to ensure that refuges, particularly those sprayed with efficacious insecticides, produce adequate numbers of susceptible alleles. Each insect/Bt crop system may have unique management requirements because of the biology of the insect, but our studies validate the need for a refuge. As we learn more about how to refine our present resistance management strategies, it is important to also develop the next generation of technology and implementation strategies.

Chromosome doubling and mode of reproduction of induced tetraploids of eastern gamagrass (Tripsacum dactyloides L.).

Eastern gamagrass, (Tripsacum dactyloides L.) is a perennial, warm-season grass that is being developed as a forage plant. Shoots were derived from callus initiated from immature embryos and immature inflorescences of diploid (2n=2x=36) gynomonoecious eastern gamagrass. These shoots were induced to microtiller in the presence of 3 mg/l benzyladenine. Amiprophosmethyl (10, 15, or 20 µM) was applied to 27 microtillers for 3-5 days to induce chromosome doubling. All 14 surviving plants were tetraploid, (2n=4x=72), as determined by flow cytometry or chromosome counts. These plants were morphologically normal and produced seed. Test crosses were made with a known diploid. Flow cytometry and chromosome counts showed that the progeny were triploid, proving that the induced tetraploids reproduce sexually.

Transgenic tobacco cell cultures expressing a Trichoderma harzianum endochitinase gene release the enzyme into the medium.

Calli and cell suspensions were obtained from tobacco plants transformed with an endochitinase-encoding cDNA from the biocontrol fungus Trichoderma harzianum. Calli from four primary transformants had high levels of endochitinase activity, like the plants from which they were derived. Endochitinase activity was also detected in the medium surrounding the calli and in the medium from transgenic cell suspensions. Western blots demonstrated the presence of the expected 40-kDa T. harzianum protein in transgenic samples but not in controls. These results indicate that the fungal enzyme is secreted and that the fungal signal peptide in the cDNA construct functions in plant cells. A cell suspension medium in which the protein concentration was increased up to 34-fold by ammonium sulfate precipitation inhibited germination of Penicillium digitatum spores. Some inhibition of spore germination was also observed in concentrated medium from control suspensions, probably due to the secretion and concentration of endogenous enzymes.

Control of Lepidopteran insect pests in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) transformed with a synthetic Bacillus thuringiensis cry1C gene.

A synthetic Bacillus thuringiensis cry1C gene was transferred to three Korean cultivars of Chinese cabbage via Agrobacterium tumefaciens-mediated transformation of hypocotyl explants. Hygromycin resistance served as an efficient selective marker. The transformation efficiency ranged from 5% to 9%. Transformation was confirmed by Southern blot analysis, PCR, Northern analysis, and progeny tests. Many transgenic plants of the closed-head types (lines Olympic and Samjin) flowered in vitro. Over 50 hygromycin-resistant plants were successfully transferred to soil. The transgenic plants and their progeny were resistant to diamondback moths (DBM, Plutella xylostella), the major insect pest of crucifers world-wide, as well as to cabbage loopers (Trichoplusia ni) and imported cabbage worms (Pieris rapae). Both susceptible Geneva DBM and a DBM population resistant to Cry1A protein were controlled by the Cry1C-transgenic plants. The efficient and reproducible transformation system described may be useful for the transfer of other agriculturally important genes into Chinese cabbage.

Ultrastructural effects of Helminthosporium maydis race T toxin on mitochondria of corn roots and protoplasts.

Zea mays inbred W64A in Texas (T, toxin sensitive) male sterile and non-male sterile (N, toxin resistant) cytoplasms were utilized. Roots of freshly germinated seeds were treated for 15 min of 2 hr with culture filtrate from liquid grown Helminthosporium maydis Race T, or with a chloroform extractable purified fraction from the culture filtrate. In the susceptible W64A T line, toxin treatment, both crude and purified, caused swelling and loss of matrix densiy in mitochondria of root cap and vacuolated cells in the region of elongation. One hour treatment with the chloroform extractable toxin fraction caused similar effects or mitochondria of isolated leaf protoplasts. This is the first report of such rapid in vivo effects of HmT toxin on mitochondria. Difficulty in obtaining consistent preservation of meristem mitochondria precluded drawing firm conclusions concerning that region of the root. In the resistant W64A N line, protoplast and root mitochondria were unaffected by the toxin.

Identifying sources and mechanisms of resistance in crucifers for control of cabbage maggot (Diptera: Anthomyiidae).

The cabbage maggot, Delia radicum (L.) is an important insect pest of eruciferous crops in upstate New York. This species causes considerable damage to seedlings and young plants by feeding on roots and stems, resulting in plant stand loss and yield loss. Five crucifer accessions (Brassica oleracea variety italica L.,'Green Comet'; B. oleracea L.,'Rapid Cycling' [Crucifer Genetics Cooperative 3-1 ]; B. oleracea variety botrytis L., a standard cauliflower cultivar'Amazing'; B. carinata L.; and Sinapis alba L., 'Cornell Alt 543') were evaluated to identify sources and mechanisms of resistance for D. radicum. Of the accessions tested, S. alba Cornell Alt 543 demonstrated reduced oviposition by D. radicum, reduced weights and survivorship of larvae, pupae or adults, and reduced damage to plants. Thus, S. alba Cornell Alt 543 could be a potential source for resistance to be bred into cruciferous crops for control of D. radicum.

Different cross-resistance patterns in the diamondback moth (Lepidoptera: Plutellidae) resistant to Bacillus thuringiensis toxin Cry1C.

Two strains of the diamondback moth, Plutella xylostella (L.), were selected using Cry1C protoxin and transgenic broccoli plants expressing a Cry1C toxin of Bacillus thuringiensis (Bt). Both strains were resistant to Cry1C but had different cross-resistance patterns. We used 12 Bt protoxins for cross-resistance tests, including Cry1Aa, Cry1Ab, Cry1Ac, Cry1Bb, Cry1C, Cry1D, Cry1E, Cry1F, Cry1J, Cry2Ab, Cry9Aa, and Cry9C. Compared with the unselected sister strain (BCS), the resistance ratio (BR) of one strain (BCS-Cry1C-1) to the Cry1C protoxin was 1,090-fold with high level of cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1F, and Cry1J (RR > 390-fold). The cross-resistance to Cry1A, Cry1F, and Cry1J in this strain was probably related to the Cry1A resistance gene(s) that came from the initial field population and was caused by intensive sprayings of Bt products containing Cry1A protoxins. The neonates of this strain can survive on transgenic broccoli plants expressing either Cry1Ac or Cry1C toxins. The other strain (BCS-Cry1C-2) was highly resistant to Cry1C but not cross-resistant to other Bt protoxins. The neonates of this strain can survive on transgenic broccoli expressing Cry1C toxin but not Cry1Ac toxin. The gene(s) conferring resistance to Cry1C segregates independently from Cry1Ac resistance in these strains. The toxicity of Cry1E and Cry2Ab protoxins was low to all of the three strains. The overall progress of all work has resulted in a unique model system to test the stacked genes strategy for resistance management of Bt transgenic crops.

Greenhouse tests on resistance management of Bt transgenic plants using refuge strategies.

Experimental evaluation of the effectiveness of resistance management tactics is vital to help provide guidelines for the deployment of transgenic insecticidal crops. Transgenic broccoli expressing a Cry1Ac gene of Bacillus thuringiensis (Bt) and the diamondback moth, Plutella xylostella (L.), were used in greenhouse tests to evaluate the influence of size and placement of nontransgenic refuge plants on changes in resistance allele frequency and pest population growth. In the first test with an initial Cry1Ac-resistance (R) allele frequency of 0.007, P. xylostella were introduced into cages with the following treatments: 0, 3.3, 10, 20, and 100% refuge plants. Results after four generations showed that resistance could be delayed by increasing the proportion of refuge plants in the cage. Population growth was also influenced by refuge size with the highest populations occurring in treatments that had either no refuge plants or all refuge plants. In the second test, we evaluated the effect of refuge placement by comparing 20% separate and 20% mixed refuges. P. xylostella with an initial frequency of resistant alleles at 0.0125 were introduced into cages and allowed to cycle; later generations were evaluated for resistance and population growth. Separating the refuge had a pronounced effect on delaying resistance and slowing establishment of resistant larvae on Bt plants. Combining information from both trials, we found a strong negative correlation between the number of larvae on Bt plants and the mortality of the population in leaf dip bioassays. Results from larval movement studies showed that separate refuges delayed resistance better than mixed refuges because they conserved relatively more susceptible alleles than R alleles and did not increase the effective dominance of resistance.

Trichoderma harzianum Endochitinase Does Not Provide Resistance to Meloidogyne hapla in Transgenic Tobacco.

Eggs of Meloidogyne hapla contain chitin, a substrate for chitinase. Our goal was to determine if endochitinase from the biocontrol fungus T. harzianum expressed in transgenic tobacco increases resistance to this nematode. Endochitinase-transgenic T tobacco seedlings expressing increased endochitinase activity in leaves (11 to 125 times over control) and roots (2 to 15 times over control) were transferred to quartz sand:loam soil mix (4:1 ratio) and inoculated with 5,000 M. hapla eggs/pot. Tomato (cv. Rutgers), pepper (cv. California Dream), and non-transformed tobacco plants were used as susceptible controls. Two experiments were performed in the greenhouse with nine and ten transgenic tobacco lines, respectively. Roots were harvested 55 days after inoculation, and number of eggs, secondstage juveniles (J2), reproductive factor (Rf), and (eggs + nematodes [J2])/g of fresh root weight were determined. The reproduction factor for tobacco plants ranged from 1.06 to 3.40. Significant differences in number of J2 and egg counts were found between some transgenic lines and control tobacco; however, they were not consistent for lines tested in both experiments. No statistical differences were detected for (eggs + nematodes [J2])/g of fresh root weight in either experiment. We conclude that the elevated endochitinase activity did not provide protection against root-knot nematodes.

Independent measurement of the total active 8B solar neutrino flux using an array of 3He proportional counters at the Sudbury Neutrino Observatory.

The Sudbury Neutrino Observatory (SNO) used an array of 3He proportional counters to measure the rate of neutral-current interactions in heavy water and precisely determined the total active (nu_x) 8B solar neutrino flux. This technique is independent of previous methods employed by SNO. The total flux is found to be 5.54_-0.31;+0.33(stat)-0.34+0.36(syst)x10(6) cm(-2) s(-1), in agreement with previous measurements and standard solar models. A global analysis of solar and reactor neutrino results yields Deltam2=7.59_-0.21;+0.19x10(-5) eV2 and theta=34.4_-1.2;+1.3 degrees. The uncertainty on the mixing angle has been reduced from SNO's previous results.

Organellar segregation, rearrangement and recombination in protoplast fusion-derived Brassica oleracea calli.

Cauliflower protoplasts were fused to determine the effect of protoplast source and pretreatment on organellar segregation in fusion products. Mitochondrial and chloroplast type were determined for over 250 calli from eight fusions between iodoacetate-treated or γ-irradiated leaf or hypocotyl protoplasts with fertile or Ogura cytoplasms. Organelles in fusion-derived calli were identified with five mitochondrial probes and one chloroplast probe. Mitochondrial and chloroplast segregation were independent but biased. Most calli had B. oleracea chloroplasts, but more calli had Ogura mitochondria than B. oleracea ones. Neither protoplast source nor pretreatment alone affected organelle segregation. However, iodoacetate treatment of hypocotyl protoplasts reduced their mitochondrial contribution to the fusion products although it did not affect chloroplast segregation. Over half of the calli had mitochondrial genomes distinct from those of either fusion partner; many of these contained the complete mitochondrial genome of one partner along with some mitochondrial DNA from the other. Out of 258 calli, 83 showed evidence of mitochondrial recombination, most commonly by formation of a novel 11-kb PstI fragment near the atp9 region.

In vitro tuberization of potato clones from different maturity groups.

In vitro tuberization on shoot cultures of early, mid-season, late and very late potatoes was compared. Shoots were grown at 12, 16, or 20 h photoperiods; tuberization was then induced at 0, 8 or 16 h light. In the dark, shoots from early plants initially grown at 16 h consistently set tubers earlier than the other types, whereas the very late line tuberized later and produced significantly fewer tubers. Tuber setting of mid-season plants could not be distinguished from the late type. Tuberization of the very late line was significantly hastened by shortening the photoperiod from 20 h to 12 h during the shoot growth period. Light during tuber induction delayed tuberization. This system may be useful to screen callus-derived plants for maturity, and may also be suitable for in vitro study of the photoperiodic control of tuberization.

Effect of cellulases on spontaneous fusion of maize protoplasts.

The effect of protoplast-isolating enzymes on spontaneous fusion of maize protoplasts (Zea mays L. cv. Black Mexican Sweet) was investigated using a convenient ethidium bromide nuclear staining procedure. After 2-2.5 hour digestion in an enzyme solution containing 1% Cellulysin, 0.5% Rhozyme, and 0.02% Pectolyase Y-23, 50-75% of the protoplasts contained multiple nuclei. The cellulase Cellulysin was identified as the factor causing the spontaneous protoplast fusion; when Cellulysin was replaced by CELF cellulase, most protoplasts were uninucleate. Calcium and other components in the enzyme solution did not affect spontaneous fusion. Cellulysin also increased the percentage of multinucleate protoplasts from rice and asparagus suspensions. Presence of multiple nuclei might affect genetic manipulations involving protoplasts.

Stimulation of extracellular polysaccharide synthesis in oat protoplasts by the host-specific phytotoxin victorin.

The host-specific phytotoxin victorin (HV-toxin) stimulates mesophyll protoplasts of susceptible but not of resistant oat (Avena sativa L.) to produce an amorphous, ethanol-insoluble extracellular material which stains with Calcofluor white and aniline blue. Over a 24-h period incorporation of [(14)C]glucose into ethanol-insoluble products is maximally stimulated by 60 pg victorin/ml, whereas at 6 ng/ml initial rates of incorporation are higher but the protoplasts collapse. The extracellular material produced in response to victorin is solubilized by cold 4.4 N NaOH and by commercial laminarinase and pectinase. Incorporation of [(14)C]glucose into cellulose (material resistant to Updegraff's acetic-nitric acid reagent) is stimulated as much as incorporation into other wall polysaccharides, but cellulose constitutes less than 15% of the total victorin-stimulated incorporation. Synthesis of ethanol-insoluble material that can be digested by pronase, i.e. protein, is inhibited by victorin above 60 pg victorin/ml. Formation of extracellular polysaccharide is stimulated at concentrations of victorin which cause almost complete inhibition of protein synthesis, indicating that de-novo protein synthesis is not involved. Preincubation of protoplasts with inhibitors of RNA or protein synthesis prevents both extracellular polysaccharide synthesis and cell death in response to victorin. Although previous studies have indicated a link between calcium and the action of victorin, several compounds which interact with calcium do not influence this response to victorin.

Novel flowering and fatty acid characters in rapid cycling Brassica napus L. resynthesized by protoplast fusion.

Novel rapid cycling Brassica napus lines have been produced by protoplast fusion between rapid cycling B. oleracea and rapid cycling B. rapa. Fusion products were selected based on iodoacetate inactivation and regeneration ability. A total of 36 plants was recovered from 3 regenerating calli. All were confirmed as somatic hybrids by morphological features, flow cytometric estimation of nuclear DNA content, RAPD analysis and/or DNA hybridization. Plants from two of the calli contained chloroplasts from B. rapa, and plants from the third contained B. oleracea chloroplasts. Some plants flowered in vitro, but on average flowering was initiated 22 days after transfer to soil. Although seed set was fairly low after self pollination, more seeds were obtained from pollination of open flowers than from pollination of buds. Seeds of the somatic hybrid B. napus showed novel fatty acid compositions, different from the mean of the two parental lines. Flowering was monitored in plants grown from seeds of the somatic hybrids, rapid cycling B. napus (CrGC 5-1) and the two diploid parental genotypes. Progeny of the somatic hybrids flowered faster and were more vigorous than rapid cycling B. napus (CrGC 5-1). The improved lines contain chloroplasts from B. rapa, unlike rapid cycling B. napus (CrGC 5-1), which has B. oleracea chloroplasts. The somatic hybrid lines produced may be useful for genetic studies or further in vitro manipulations.

Regeneration of plants from protoplasts of rapid cycling Brassica oleracea L.

Rapid cycling Brassica species have great potential in plant genetic research because of their short life cycles and their minimal space requirements. Rapid cycling B. oleracea can be grown with up to six generations per year. Protoplast culture of this genotype can be applied for gene transfer by direct DNA uptake and by protoplast fusion. We here report on fast regeneration of flowering plants from protoplasts of rapid cycling B. oleracea. Regeneration frequencies of 27-65% were achieved with multiple shoots developing from individual calli. The regenerated plants were grown to maturity, and flowering and other morphological characteristics were monitored. The regenerants flowered within a similar time frame as plants grown from seeds. The ploidy level of regenerated and seed-grown plants was measured by flow cytometry. Many (20-45%) of the regenerants were tetraploid. Although only few seeds could be obtained from the tetraploids, large numbers of seeds with good germination were recovered from the diploid regenerants.

Direct and sensitive fluorescence in situ hybridization of 45S rDNA on tomato chromosomes.

We describe a direct and sensitive fluorescence in situ hybridization protocol for plant chromosomes. We labelled 45S rDNA with fluorescein-12-dUTP and hybridized to somatic chromosomes of four tomato genotypes. This technique does not require posthybridization immunocytochemical amplifications. The improved signal sensitivity with this technique allowed identification of new rDNA loci on three pairs of chromosomes, in addition to the previously known locus on chromosome 2. We discuss favorable features of direct fluorescence in situ hybridization for chromosomes fixed on a slide and chromosomes or cells in suspension.

Transgene expression in broccoli (Brassica oleracea var. italica) clones propagated in vitro via leaf explants.

We have developed an efficient protocol for the in vitro propagation of transgenic broccoli plants using leaf explants as starting material. A high frequency of shoot formation from leaf explants was obtained on Murashige and Skoog medium containing benzyladenine (BA, 5 mg/l) and naphthaleneacetic acid (0.5 mg/l). Frequent subcultures of existing shoots and shoot clusters to medium containing only BA (2 mg/l) promoted rapid shoot multiplication. The use of a 1:1 mixture of Agargel and Gelrite in the rooting medium increased the number of healthy roots per rooted plant. Applying this protocol, we obtained thousands of clonal rooted plantlets within 6 months from a transgenic broccoli plant carrying the cry1Ac and cry1C genes from Bacillus thuringiensis associated with kanamycin and hygromycin selectable markers, respectively. Thirty randomly selected clones that had been propagated for 1 year on medium containing kanamycin (50 mg/l) all showed resistance to both kanamycin and hygromycin. Genomic DNA and total soluble proteins were isolated from 16 of these clones. Polymerase chain reaction analysis indicated that the cry1Ac and cry1C genes were both maintained. ELISA assays showed that all of the clones produced a high level of Cry1Ac protein similar to the original transgenic plant; however, most clones had significantly lower levels of Cry1C protein than the original plant. This variation indicates that it is important to evaluate transgene expression in transgenic clones propagated long-term in vitro. In vitro propagation starting from leaf explants was also successful with other transgenic and non-transgenic Brassica oleracea materials, including broccoli, cauliflower, and collard.