Assessing the susceptibility of cruciferous Lepidoptera to Cry1Ba2 and Cry1Ca4 for future transgenic cruciferous vegetables.

Advances in transgenic plants expressing Bacillus thuringiensis (Bt) insecticidal gene(s) offer a promising alternative to traditional insecticides for control of lepidopteran pests on important cruciferous vegetable crops such as cabbage and cauliflower. A public-private partnership, the Collaboration on Insect Management for Brassicas in Asia and Africa (CIMBAA), was formed in 2005 with the goal of developing dual-gene Bt cauliflower and cabbage, initially for India, to replace the use of broad spectrum, traditional insecticides. As a first step in this effort, the major lepidopteran pests of cruciferous vegetable crops [Plutella xylostella (L.), Pieris rapae (L.), Pieris brassicae (L.), Crocidolomia binotalis (L.), Hellula undalis (F.), Diacrisia obliqua Walker, Spodoptera litura F., and Helicoverpa armigera (Hübner)] were collected over a large geographic area (India, Indonesia, Taiwan, China, Australia, and the United States) and tested against purified Cry1Ba2 and Cry1Ca4 toxins, the toxins proposed to be expressed in the CIMBAA plants. Our results demonstrate that Cry1Ba2 and Cry1Ca4 were effective against the primary target of the CIMBAA plants, P. xylostella, regardless of geographic location, and had LC50 values <1.3 ppm. Furthermore, one or both toxins were effective against the other major pest Lepidoptera, except for S. litura or H. armigera which were less susceptible. No cross-resistance has been found between Cry1Ba2 and Cry1Ca4, suggesting cry1Ba2+cry1Ca4 cauliflower and cabbage could be an effective and sustainable tool to control, P. xylostella, the key lepidopteran pest on cruciferous vegetable crops, as well as most other Lepidoptera. As the CIMBAA plants are being developed, further tests are needed to determine whether they will express these proteins at sufficient levels to control all the Lepidoptera. Sustainable use of the dual-gene plants also is discussed.

Plastid transformants of tomato selected using mutations affecting ribosome structure.

Tomato plastid transformants were obtained using two vectors containing cloned plastid DNA of either Nicotiana tabacum or Solanum nigrum and including point mutations conferring resistance to spectinomycin and streptomycin. Transformants were recovered after PEG-mediated direct DNA uptake into protoplasts, followed by selection on spectinomycin-containing medium. Sixteen lines contained the point mutation, as confirmed by mapping restriction enzyme sites. One line obtained with each vector was analysed in more detail, in comparison with a spontaneous spectinomycin-resistant mutant. Integration of the cloned Solanum or Nicotiana plastid DNA, by multiple recombination events, into the tomato plastome was confirmed by sequence analysis of the targeted region of plastid DNA in the inverted repeat region. Maternal inheritance of spectinomycin and streptomycin resistances or sensitivity in seedlings also confirmed the transplastomic status of the two transformants. The results demonstrate the efficacy in tomato of a selection strategy which avoids the integration of a dominant bacterial antibiotic resistance gene.

Training for more accurate visual fat estimation in meat.

Much animal fat in the diet is contained in meat. As fat intake is considered too high in western societies, a more fat-conscious attitude may be desirable. One of the parties involved is the butcher, who sells fresh meat directly to the consumer. In a pre-post experimental design, with an interpolated training phase, the possibility to improve the ability of student butchers to visually estimate fat content of meat, was investigated. A limited number of training sessions, in which immediate feed-back was given of the actual fat percentage after each estimation, led to a large improvement in fat estimation accuracy. A delayed post-test indicated that most of the training effect was preserved after six weeks. Similarities between the observed learning process and informational feed-back learning with numerosity stimuli were discussed. On the basis of these results it is recommended that courses for trainee butchers include a short course on fat estimation in their curriculum. If butchers sell what they think they sell, consumers are more likely to get what they think they get. Increased 'fat awareness' may indirectly contribute to healthier eating habits.

Inhibition by indomethacin and 5,8,11,14-eicosatetraynoic acid of the induction of rat hepatic ornithine decarboxylase by the tumor promoters phenobarbital and 12-O-tetradecanoylphorbol-13-acetate in vivo.

The involvement of arachidonate metabolism in the induction of rat hepatic ornithine decarboxylase (ODC) activity by the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phenobarbital (PB) was investigated. Pretreatment of the rats with indomethacin or 5,8,11,14-eicosatetraynoic acid dose dependently inhibited the induction of ODC by both tumor promoters. Both inhibitors were more potent inhibitors of PB induction than TPA induction of ODC. The data are consistent with an involvement of arachidonate cyclooxygenase products in the induction of rat hepatic ODC by the tumor promoters.

Production of potato monohaploids (2n=x=12) through prickle pollination.

Data are presented on the potential of gynogenesis for the production of monohaploids and on factors affecting their frequency and relative vigour. Diploid Solanum tuberosum L. and S. tuberosumxS. phureja Juz et Buk hybrids were used as maternal parents and selected S. phureja clones as prickle pollinators with embryo-spot and nodal band as dominant seed and plant marker. About 2 million seeds were screened for absence of embryo-spot. After raising plants from phenotypically spotless seeds, further screening for absence of nodal bands and for ploidy level was carried out. Finally more than 500 monohaploid plants from three genetically different groups of maternal parents were obtained. Frequency and vigour of the monohaploids were clearly dependent on their maternal genotypes. The data also indicated an effect of the pollinator genotype, the physiological stage of the maternal plant and the environment on monohaploid frequency. On the basis of these results the possibility of breeding for a higher monohaploid production rate and for more stable and vigorous monohaploids is discussed. Furthermore, gynogenesis and androgenesis are compared. It is suggested that both should be used in order to obtain monohaploids from sufficiently various diploid breeding material.

Relative performance of monohaploid potato clones and their diploid parents at plant level and after protoplast isolation and subsequent fusion.

Plant growth performance was studied in 118 potato monohaploids and in their diploid parents. Of these monohaploids 76 were also investigated at the protoplast level and eight of these were used in protoplast fusion experiments as well. No correlation was found between relative performance of greenhouse grown and in vitro grown plants. No or only weak correlations were found between different in vitro characteristics such as plant growth, protoplast yield per gram plant material, plating efficiency and callus growth. This indicates the unpredictability of these characters.The protoplast fusion experiments indicated that only in some genotype combinations increased callus growth rates may be found. However, it is not clear whether such calli were hybrids or not. In protoplast monocultures only diploid and tetraploid regenerants were obtained. After fusion, tetraploids but also some triploids could be regenerated. The finding of triploids indicates that monoploid protoplasts were involved in fusion. Isozyme analysis and morphological assessment of the plants pointed out that the majority of the fusion regenerants were hybrids. The implications of these results are discussed.

Ploidy variability in greenhouse cultured and in vitro propagated potato (Solanum tuberosum) monohaploids (2n=x=12) as determined by flow cytometry.

The DNA distributions of 23 different monohaploid potato clones were investigated by flow-cytometric measurements. All monohaploid clones differed in DNA distribution but none of them contained only monoploid cells in the leaves. All were highly stable on the monohaploid level for 2-3 years. Investigation of the influence of different factors on the DNA distribution in leaf cells showed that the material derived from in vitro shoot tip propagation contained a lower proportion of polyploidized cells than greenhouse grown plants. With protoplast isolation the enzyme treatment of in vitro cultured plant material induced a striking shift of DNA distribution towards the lower C-value whereas the mechanical purification steps caused a selective loss of monoploid nuclei. Seasonal influence on the DNA patterns could be detected.

Effect on hepatic ornithine decarboxylase of some food additives and synthetic elastomers.

The food additives butylated hydroxytoluene, butylated hydroxyanisole and sodium cyclamate and the precursors for packaging materials acrylamide, acrylic acid, acrylonitrile and vinylpyrrolidone were investigated for their ability to induce hepatic ornithine decarboxylase (ODC) in vivo, in order to obtain indications about their possible tumour-promoting activities. It was shown that butylated hydroxyanisole, acrylonitrile, vinylpyrrolidone and acrylamide have the capacity to increase rat liver ODC activity, while butylated hydroxytoluene, acrylic acid and sodium cyclamate did not affect ODC activity.

Effect of 1,8-dihydroxy-9-anthrone (anthralin) on rat hepatic ornithine decarboxylase activity in vivo.

Intraperitoneal injection of the non-phorbol tumor promoter anthralin (1,8-dihydroxy-9-anthrone) in male rats resulted in an increase of hepatic ornithine decarboxylase (ODC) activity. Maximal activity was observed 8 h after promoter administration reaching levels about 30 times over control. The kinetics of anthralin dependent ODC induction differed markedly from that by either 12-O-tetradecanoylphorbol-13-acetate (TPA) or phenobarbital (PB) (Bisschop et al., Carcinogenesis 2 (1981) 1282). With anthralin a slow decrease of ODC back to control level is observed approximately within 22 h. In contrast, ODC induction mediated by other tumor promoters like TPA and PB decreased to control levels within 4-6 hours. Administration of a second dose of anthralin 8 h after the first dose prevented the activity decrease as normally observed after a single dose of a tumor promoter. This effect lasted at least 10 h. ODC activity induction occurred in a dose-dependent manner being linear from 10-2000 micrograms anthralin/kg body wt. Pretreatment of the animals either with actinomycin D or with cycloheximide completely blocked anthralin mediated ODC induction suggesting that de novo ODC-mRNA synthesis and subsequent translation is involved in this process.