RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM–mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.

To produce the homozygous strain of a haploid plant derived from small seed-derived seedlings of `Banpeiyu' pummelo (Citrus grandis Osbeck), we carried out colchicine treatment to axillary shoot buds of the haploid. Many shoots with cytochimeras (X+2X and 2X+4X) arose from the colchicine-treated axillary buds. When cytochimeric buds of 2X+4X were top-grafted onto trifoliate orange [Poncirus trifoliata (L.) Raf.], a complete diploid shoot with 18 chromosomes was obtained from the cytochimera. This diploid strain showed vigorous growth compared with the original haploid. The leaf weight per unit area and the stomata size in this diploid were significantly larger than those of the original haploid plant, and were almost equal to those of `Banpeiyu' pummelo. The diploid strain was confirmed to be a doubled haploid of a haploid from `Banpeiyu' pummelo, based on random amplified polymorphic DNA (RAPD) analysis and chromosome composition analysis by chromomycin A3 (CMA) staining.

Embryogenic cell suspension cultures were established using the ovule culture of an interspecific cross, Alstroemeria pelegrina var. rosea × A. magenta. Ovules harvested 14 d after pollination were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGRs); calli were produced on the hypocotyl surface in germinating zygotic embryos. Suspension cells were induced from the calli by using liquid MS media containing 2,4-dichlorophenoxyacetic acid or 4-amino-3,5,6-trichloropyridine-2-carboxylic acid (picloram). Adventitious embryos developed from the suspension cells on half-strength MS medium supplemented with 0.5 mg l -1 of both α-naphthaleneacetic acid and N 6 -benzylaminopurine; they grew into 2 plantlets on the same medium. The plantlets formed rhizomes following transfer to half-strength MS medium without PGRs, and acclimatized plants were easily established. Subsequently, Agrobacterium-mediated transformation system was applied.The suspension cells were co-cultivated with A. tumefaciens strain EHA101/pIG121Hm or LBA4404/pTOK233, both of which contain neomycin phosphotransferase II, hygromycin phosphotransferase and intron-containing ß-glucuronidase (intron-GUS)genes. Seven days after co-cultivation, the cells were subjected to GUS assay; staining

In the present study, in vitro ovule culture technique was used to obtain interspecific cross combination of Dioscorea rotundata ufenyi and Dioscorea bulbifera wild. Ten days after pollination, ovules were excised and cultured onto 1/2 strength Murashige and Skoog (MS) medium (Basal salt mixture + Vitamins) supplemented with 6% sucrose, 0.7% agar and plant growth hormones such as GA3, BAP, Picrolam and TDZ. Cultured ovules were transferred on 1/2 MS medium with 3% sucrose and 0.7% agar after three weeks. 40 days after pollination, germination was observed from 7 months cultured ovule between D. rotundata ufenyi x D. bulbifera wild. Hybridity of the regenerated plant was checked by flow cytometric method. A close relation was observed between the fluorescence intensity of the obtained progeny with one of the parents' fluorescence. The observed progeny can be closely correlated with an apomictic tissue from an ovule parent of D. rotundata ufenyi. Plantlets derived from ovule culture were proliferated through in vitro shoot multiplication with hormonal concentration (0.5 mg/l BAP) supplemented with 1/2 strength MS medium. Obtained ovule culture derived in vitro plantlets were successfully hardened, acclimatized and transferred to the field, where they survived and grew normally. In plant breeding, interspecific crossing is very important technique, enabling the time needed to produce homozygous lines to be shortened as compared to the conventional plant breeding techniques.

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