A synthetic Bacillus thuringiensis (Bt) cry1C gene under the control of the 35S CaMV promoter was introduced into cauliflower (Brassica oleracea var. botrytis) by Agrobacterium tumefaciens- mediated transformation with hygromycin selection. A total of 35 transgenic plants were regenerated from six cultivars (Freemont, Candid Charm, Snow Crown, Cumberland, Majestic, and Cashmere) with average transformation efficiency of 0.3% to 6.4%. All the hygromycin-resistant transformants also carried the Bt gene, as shown by PCR with primers specific to the cry1C gene. ELISA analysis showed that the levels of Cry1C protein in independent transformants varied widely, from 0 to 0.2% of total soluble protein. The majority of the plants (61%) produced a high level of Cry1C protein (> 1000 ng mg_1 proteins). Insect bioassays demonstrated that plants producing Cry1C protein effectively controlled larvae of diamondback moths (Plutella xylostella), including ones resistant to Cry1A protein, as well as larvae of cabbage loopers (Trichoplusia ni). These cry1C cauliflower plants will be useful for further studies, especially in comparisons with cauliflower plants carrying the same cry1c gene under control of a light-inducible promoter.

The 'Anand' type of cytoplasmic male sterility (CMS) was introduced to Brassica oleracea through protoplast fusion (Cardi and Earle, 1997), providing a source of male sterility that is of value in producing F1 hybrid varieties of this species without the cold sensitivity of 'Ogura' cytoplasm. In this species, as in others in which it has been introduced, 'Anand' mitochondria are associated with flower abnormalities. The best lines have been used by commercial breeders. This paper describes 'Anand' flower abnormalities in further detail. Rapid cycling brassica with 'Anand' cytoplasm may have promise as a model system for research on floral patterning. Materials and Methods. Three lines of rapid cycling B. oleracea carrying the 'Anand' cytoplasm and one wild type were grown. The flower morphology of 'Anand' lines was classified as good in F25-F-1A, fair in F25-F-6B, and poor in F23-F-1B (Cardi and Earle, 1997). Seedlings were started in an indoor growth room, but were maintained in a naturally lit greenhouse at 20° night, 25° day during flowering. Five flowers on 5 plants of each line were examined. Results. Morphological abnormalities affected all floral parts (Table 1). There was substantial variation among flowers on individual plants and among lines but little within lines. Conclusions. "Good" flowers have large petals that push open the sepals before the carpel elongates. Female fertility could be reduced due to occlusion of the stigma or physical damage caused by the stigma pressing against the end of a sepal and kinking the style. Female fertility in some flowers was unlikely because the carpel suture failed to fuse. In some cases the edge of the split carpel was sepal-like, consistent with the absence of SEPALLATA expression. The stigma on a sepal represents the converse. Stamens were most strongly affected, with outer stamens most reduced. This whorl is the one most frequently suppressed in flowering plants (Masters, 1869). The number of stamens was reduced to varying degrees. First to be lost was one or both of the pair outside the petals. Then the outer pair inside the petals was affected. One or both of these stamens could either be lost or doubled to give 3 to 5 stamens. Again a positional cue for stamens appears to be weakened. Anther development was often limited, the expected and desired consequence of the Anand genotype. Some undehisced " good " anthers contained pollen. There is variation even among the lines initially described as " good ". Further selection among these may help identify those that are most promising for further use in hybrid production.

Understanding the genetic relationships among farmer-preferred cassava (Manihot esculenta Crantz) varieties is indispensable to genetic improvement efforts. In this study, we present a genetic analysis of 547 samples of cassava grown by 192 smallholder farmers, which were sampled at random within four districts in Uganda. We genotyped these samples at 287,952 single nucleotide polymorphisms using genotyping-by-sequencing and co-analyzed them with 349 cassava samples from the national breeding program in Uganda. The samples collected from smallholders consisted of 86 genetically unique varieties, as assessed using a genetic distance-based approach. Of these varieties, most were cultivated in only one district (30 in Kibaale, 19 in Masindi, 14 in Arua, and three in Apac), and only three were cultivated across all districts. The genetic differentiation we observed among farming districts in Uganda (mean fixation index [F-ST] = .003) is similar to divergence observed within other countries. Despite the fact that none of the breeding lines were directly observed in farmer fields, genetic divergence between the populations was low (F-ST = .020). Interestingly, we detected the presence of introgressions from the wild relative M. glaziovii Mull. Arg. on chromosomes 1 and 4, which implies ancestry with cassava breeding lines. Given the apparently similar pool of alleles in the breeding germplasm, it is likely that breeders have the raw genetic material they require to match the farmer-preferred trait combinations necessary for adoption. Our study highlights the importance of understanding the genetic makeup of cassava currently grown by smallholder farmers and relative to that of plant breeding germplasm.

We investigated whether development of resistance to a Bt crop in the presence of a natural enemy would be slower than without the natural enemy and whether biological control, in conjunction with a Bt crop, could effectively suppress the pest population. Additionally, we investigated whether insecticide-sprayed refuges of non-Bt crops would delay or accelerate resistance to the Bt crop. We used a system of Bt broccoli expressing Cry1Ac, a population of the pest Plutella xylostella with a low frequency of individuals resistant to Cry1Ac and the insecticide spinosad, and a natural enemy, Coleomegilla maculata, to conduct experiments over multiple generations. The results demonstrated that after 6 generations P. xylostella populations were very low in the treatment containing C. maculata and unsprayed non-Bt refuge plants. Furthermore, resistance to Bt plants evolved significantly slower in this treatment. In contrast, Bt plants with no refuge were completely defoliated in treatments without C. maculata after 4-5 generations. In the treatment containing sprayed non-Bt refuge plants and C. maculata, the P. xylostella population was low, although the speed of resistance selection to Cry1Ac was significantly increased. These data demonstrate that natural enemies can delay resistance to Bt plants and have significant implications for integrated pest management (IPM) with Bt crops.

The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Lepidoptera on cotton and maize and risk assessment studies are needed to ensure they do not harm important natural enemies. However, using Cry protein susceptible hosts as prey often compromises such studies. To avoid this problem we utilized pest Lepidoptera, cabbage looper (Trichoplusia ni) and fall armyworm (Spodoptera frugiperda), that were resistant to Cry1Ac produced in Bt broccoli (T. ni), Cry1Ac/Cry2Ab produced in Bt cotton (T. ni), and Cry1F produced in Bt maize (S. frugiperda). Larvae of these species were fed Bt plants or non-Bt plants and then exposed to predaceous larvae of the green lacewing Chrysoperla rufilabris. Fitness parameters (larval survival, development time, fecundity and egg hatch) of C. rufilabris were assessed over two generations. There were no differences in any of the fitness parameters regardless if C. rufilabris consumed prey (T. ni or S. frugiperda) that had consumed Bt or non-Bt plants. Additional studies confirmed that the prey contained bioactive Cry proteins when they were consumed by the predator. These studies confirm that Cry1Ac, Cry2Ab and Cry1F do not pose a hazard to the important predator C. rufilabris. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms.

Production of double haploid (DH) lines is a rapid method of obtaining completely homozygous inbred lines for numerous crops; however, practical trials testing the use of DH onion lines have been limited. DH onion lines were produced from diverse highly heterozygous material in development within the Cornell onion breeding program. These DH lines were evaluated in multiple replicated trials in onion fields in New York as lines and as parents of hybrids to assess the impact and commercial potential of DH onions. Twenty DH onion lines were compared with open-pollinated cultivars developed from the same source germplasm and with a commercial hybrid over two years. The vegetative vigor of the DH lines was comparable to that of related open-pollinated cultivars, showing minimal, if any, inbreeding depression. Two sets of hybrids were produced using the DH lines as males and two different females that are the female parents of the commercial hybrid controls. Therefore, hybrids in each set are half-sibs, and any performance differences are attributable to their DH male lines. In four replicated trials of these hybrids and controls, the experimental hybrids were either not significantly different or significantly better in measures of vegetative vigor compared with conventional half-sib hybrid controls. The vegetative vigor of DH lines, and their derived hybrids, might result from selection of plantlets without deleterious sublethal genes during gynogenesis. The shortened development time and equivalent quality of DH lines compared with the traditionally bred onion inbreds warrant their use. The increased vigor in hybrid combination could be an additional benefit for onion breeding strategies.

Transcription of the centromeric regions has been reported to occur in G1 and S phase in different species. Here, we investigate whether transcription also occurs and plays a functional role at the mammalian centromere during mitosis. We show the presence of actively transcribing RNA polymerase II (RNAPII) and its associated transcription factors, coupled with the production of centromere satellite transcripts at the mitotic kinetochore. Specific inhibition of RNAPII activity during mitosis leads to a decrease in centromeric α-satellite transcription and a concomitant increase in anaphase-lagging cells, with the lagging chromosomes showing reduced centromere protein C binding. These findings demonstrate an essential role of RNAPII in the transcription of α-satellite DNA, binding of centromere protein C, and the proper functioning of the mitotic kinetochore.

Crop plants encounter constant biotic challenges, and these challenges have historically been best managed with resistance (R) genes. However, the rapid evolution of new pathogenic strains along with the nonavailability or nonidentification of R genes in cultivated crop species against a large number of plant pathogens have led researchers to think beyond R genes. Biotechnological tools have shown promise in dealing with such challenges. Technologies such as transgenerational plant immunity, interspecies transfer of pattern recognition receptors (PRRs), pathogen-derived resistance (PDR), gene regulation, and expression of antimicrobial peptides (AMPs) in host plants from other plant species have led to enhanced disease resistance and increased food security.

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), a major pest of cruciferous crops throughout the world, has demonstrated an ability to develop resistance to many different classes of insecticides, including proteins from Bacillus thuringiensis that are expressed in plants (Bt plants). The ovipositional preferences and larval survival of strains (resistant strain, RR; heterozygous strain, RS; susceptible strain, SS) of P. xylostella to Cry1Ac-expressing broccoli or broccoli plants treated with lambda-cyhalothrin or spinosad were studied under greenhouse condition. Numbers of eggs per plant did not differ between Bt broccoli and non-Bt broccoli for Bt-RR, Bt-RS, and Bt-SS adults. Ovipositing adults (spinosad-RR, spinosad-RS, and spinosad-SS) also could not discriminate between spinosad-treated and untreated plants, and oviposition did not increase over the 13 d after spinosad treatment. For broccoli treated with lambda-cyhalothrin at the diagnostic dose of 20 ppm, all three insect strains (lc-RR, lc-RS, and lc-SS) had constant oviposition over time based on linear regressions. At the field dose of 80 ppm, the lc-RR strain had constant oviposition over time. The lc-SS susceptible strain had increasing oviposition over time, but the oviposition pattern on the nonsprayed broccoli also increased over time. Susceptible females layed fewer eggs on plants sprayed with lambda-cyhalothrin than on unsprayed plants. A residue-persistence test showed that spinosad and lambda-cyhalothrin could effectively control SS P. xylostella larvae for 7-9 d after application. These results are discussed in relation to their potential impact on insecticide resistance management strategies.

Recovery of doubled haploid (DH) progeny from haploid melon plants for use in breeding programs requires efficient chromosome doubling procedures. We describe improved procedures for recovery of fruits and viable seeds from parthenogenetic melon plants. Plant regeneration from nodal explants treated with 500 mg/L colchicine for 12 h was increased from 40 to 88% by transferring the treated explants to medium supplemented with a combination of growth regulators [5 μM IAA; 5 μM BA; 1 μM ABA; 30 μM AgNO3). Prolonged exposure (2–7 days) to colchicine inhibited regeneration from nodal explants but had less effect on shoot tip explants. Many colchicine-treated plantlets flowered in vitro, allowing early assessment of their male fertility. Production of stained pollen in plants from nodal explants was highest after 0.5–2 days of colchicine treatment and on plants from shoot tips after 1–2 days. In vitro pollen counts correlated well with counts from greenhouse grown plants and with fruit set. The fruit set rate for colchicine-treated plants with a high pollen number was 47%. Appropriate colchicine treatment and culture of nodal explants as well as tip explants can substantially increase the number of fertile plants and DH lines recovered from parthenogenetic melons.

Little is known about the telomere chromatin dynamics of embryonic stem (ES) cell. Here, we demonstrate localization of histone H3.3 at interphase telomeres and enrichment of Ser31-phosphorylated H3.3 at metaphase telomeres in pluripotent mouse ES cells. Upon differentiation, telomeric H3.3S31P signal decreases, accompanied by increased association of heterochromatin repressive marks and decreased micrococcal nuclease sensitivity at the telomeres. H3.3 is recruited to the telomeres at late S/G2 phase, coinciding with telomere replication and processing. RNAi-depletion of H3.3 induces telomere-dysfunction phenotype, providing evidence for a role of H3.3 in the regulation of telomere chromatin integrity in ES cells. The distinctive changes in H3.3 distribution suggests the existence of a unique and functionally essential telomere chromatin in ES cells that undergoes dynamic differentiation-dependent remodeling during the process of differentiation.

Vegetable Indian mustard (Brassica juncea cv. "Green Wave") plants that control Plutella xylostella (diamondback moth) (DBM) were produced by introduction of one or two Bacillus thuringiensis (Bt) genes. A cry1Ac Bt gene associated with the nptII gene for kanamycin selection or a cry1C Bt gene with the hpt gene for hygromycin selection was introduced individually through Agrobacterium-mediated transformation of seedling explants. A cry1C line was then transformed with the cry1Ac gene to produce pyramided cry1Ac + cry1C plants. Sixteen cry1C, five cry1Ac, and six cry1Ac + cry1C plants were produced. PCR and Southern analyses confirmed the presence of the cry1C, cry1Ac or pyramided cry1Ac + cry1C genes in the Indian mustard genome. ELISA analysis showed that production of Bt proteins varied greatly among individual transgenic plants, ranging from undetectable to over 1,000 ng Bt/mg total soluble protein. The levels of the Bt proteins were correlated with the effectiveness of control of diamondback moth (DBM) larvae. Insect bioassays indicated that both the cry1C and cry1Ac plants were toxic to susceptible DBM. The cry1C plants also controlled Cry1A-resistant DBM while cry1Ac plants controlled Cry1C-resistant DBM, and the pyramided cry1Ac + cry1C plants effectively controlled all three types of DBM. These Bt-transgenic plants could be used either for direct control of DBM and other lepidopteran insect pests or for tests of "dead-end" trap crops as protection of high value non-transgenic crucifer vegetables such as cabbage.

Several types of trap crops have been recommended for managing the diamondback moth, Plutella xylostella, including collards (Brassica oleracea var. acephala) and Indian mustard (Brassica juncea L.). However, results have been variable perhaps because populations of P. xylostella develop on these trap crops and spill over to the cash crop. To overcome this problem, we sought to develop "dead-end" trap crops that were more attractive for oviposition than the cash crop but on which P. xylostella larvae cannot survive. We have produced Bacillus thuringiensis (Bt)-transgenic collard and Indian mustard lines with a cry1C gene that have the potential to be used as a "dead-end" trap crop for P. xylostella. Greenhouse and small cage studies confirmed the control of P. xylostella larvae on the Bt crops. Furthermore, Indian mustard was significantly preferred over cabbage and collards for oviposition, regardless of whether the Indian mustard was Bt or non-Bt. The use of Bt Indian mustard as a trap crop significantly reduced the number of larvae that appeared on a cabbage cash crop, compared with using a non-Bt Indian mustard trap crop. However, this reduction also occurred when using Bt collards as a trap crop, despite collards being less preferred for oviposition. In fact, despite the overall increase in oviposition caused by the presence of Indian mustard compared with collards, the use of either Bt Indian mustard or Bt collards provided the same level of protection to the cash crop. Both plants also resulted in significant suppression of a P. xylostella population over 3 generations in the greenhouse test and 2 generations in the small cage experiment, suggesting that in places where immigration may be limited some long-term population suppression may occur. We suggest that Bt trap crops may be useful tools in situations where the cash crop may not be suitable or desirable for genetic engineering.

Haploid/doubled haploid (DH) technology can aid plant breeding programs by accelerating production of homozygous lines, provided enough viable DH progeny can be obtained from diverse haploid genotypes. In cases where there is a low frequency of spontaneous doubling, chromosome doubling procedures are required to achieve fecundity. We produced 63 parthenogenetic melon plantlets via pollination with γ-irradiated pollen, cloned them by nodal cuttings, and tested the effects of in vitro and in vivo colchicine treatment on survival, ploidy, pollen production, and fruit recovery. The most effective procedure was in vitro exposure of 3 cm shoot tip explants to 500 mg/l colchicine for 3 h. This treatment gave 83% survival of explants and 26% conversion to diploidy. Fruit recovery rate was 60% among plants with good pollen production. In vivo exposure of the tops of young plants to 5000 mg/l for 2 and 4 h yielded some fruits but also resulted in less survival and more morphological abnormalities. Strategies for recovery of progeny from parthenogenetic melon plants are recommended. To our knowledge, this study represents the first comprehensive study of recovery of fruits and viable seeds from parthenogenetic melon plants.

The ecological safety of transgenic insecticidal plants expressing crystal proteins (Cry toxins) from the bacterium Bacillus thuringiensis (Bt) continues to be debated. Much of the debate has focused on nontarget organisms, especially predators and parasitoids that help control populations of pest insects in many crops. Although many studies have been conducted on predators, few reports have examined parasitoids but some of them have reported negative impacts. None of the previous reports were able to clearly characterize the cause of the negative impact. In order to provide a critical assessment, we used a novel paradigm consisting of a strain of the insect pest, Plutella xylostella (herbivore), resistant to Cry1C and allowed it to feed on Bt plants and then become parasitized by Diadegma insulare, an important endoparasitoid of P. xylostella. Our results indicated that the parasitoid was exposed to a biologically active form of the Cy1C protein while in the host but was not harmed by such exposure. Parallel studies conducted with several commonly used insecticides indicated they significantly reduced parasitism rates on strains of P. xylostella resistant to these insecticides. These results provide the first clear evidence of the lack of hazard to a parasitoid by a Bt plant, compared to traditional insecticides, and describe a test to rigorously evaluate the risks Bt plants pose to predators and parasitoids.

Transgenic brassica crops producing insecticidal proteins from Bacillus thuringiensis (Bt) are being investigated as candidates for field release to control lepidopteran pests. Information on the potential impact of Bt brassica crops on pests and non-target natural enemies is needed as part of an environmental risk assessment prior to the commercial release. This first tier study provides insight into the tritrophic interactions among Bt broccoli plants, the herbivore Pieris rapae and its parasitoid Pteromalus puparum. We first evaluated the efficacy of three types of Bt broccoli plants, cry1Ac, cry1C and cry1Ac + cry1C, on different instars of P. rapae. Bt broccoli effectively controlled P. rapae larvae, although later instars were more tolerant. The efficacy of different Bt broccoli plants on P. rapae larvae was consistently cry1Ac > cry1Ac + cry1C > cry1C. When the parasitoid P. puparum developed in a P. rapae pupa (host) that had developed from Bt plant-fed older larvae, developmental time, total number and longevity of the P. puparum generated from the Bt plant-fed host were significantly affected compared with those generated from the non-Bt control plant-fed host. Simultaneously, negative effects on P. rapae pupae were found, i.e. pupal length, width and weight were significantly reduced after older P. rapae larvae fed on different Bt plants for 1 or 2 days. Cry1C toxin was detected using ELISA in P. rapae pupae after older larvae fed on cry1C broccoli. However, no Cry1C toxin was detected in newly emerged P. puparum adults developing in Bt-fed hosts. Only a trace amount of toxin was detected from entire P. puparum pupae dissected from the Bt plant-fed host. Moreover, no negative effect was found on the progeny of P. puparum developing from the Bt plant-fed host when subsequently supplied with a healthy host, P. rapae pupae. The reduced quality of the host appears to be the only reason for the observed deleterious effects on P. puparum. Our data suggest that the effects on P. puparum developing in Bt plant-fed P. rapae are mediated by host quality rather than by direct toxicity.

Successful application of doubled-haploid (DH) technology in onion (Allium cepa L.) breeding programs requires production of large numbers of fertile DH plants. Only a small percentage of the onion plants recovered via gynogenesis are spontaneous DH, so induction of chromosome doubling is required. The chromosome doubling step is currently one of the major limitations in onion DH programs. To address this problem, three complementary strategies for increasing the frequency of DH onion plants obtained via gynogenesis were investigated. First, the doubling efficiencies of three anti-mitotic agents (amiprofos methyl (APM), colchicine, and oryzalin) on whole basal explants from in vitro haploid plant were tested. APM at 100 and 150μM and colchicine at 750 and 1000μM gave similar regeneration of explants (70–80%) and recovery of diploid plants (25–32%). Colchicine is highly toxic to mammalian cells, so the comparable efficacy of APM at much lower concentrations offers a safer approach. Some gynogenic plants remain haploid even after exposure to anti-mitotic agents. The second strategy recovered diploid plants from such haploid plants via spontaneous chromosome doubling in somatic shoots regenerated from haploid flowers cultured in vitro. About 60% of the somatic shoots formed on flowers from haploid plants were diploid. Addition of colchicine (12.5–50μM) to the shoot regeneration medium did not increase the frequency of diploid plants recovered but raised the frequency of tetraploid somatic plants. The third strategy used a second cycle of gynogenesis to recover diploid plants from in vivo tetraploid and mixoploid plant materials. The diploid gynogenic plants recovered produced bulbs comparable to those of diploid control plants, averaging ca. 200g. Use of the three strategies in combination can maximize the recovery of fecund DH plants in gynogenesis-derived populations for use in onion breeding programs.