[Detection and analysis of alien genes in transgenic rice by fluorescence in situ hybridization].

Using fluorescence in situ hybridization (FISH) with metaphase preparations, we localized a transferred barnase-psl DNA sequence onto chromosomes in 8 rice transgenic plants. All the tested rice transgenic lines showed hybridization signals on the middle and terminal regions of chromosome arms except for those close to centromeres. In two lines, two different integration sites were identified, and the other lines showed only one integration site. With the aid of Southern analysis and expression detection, we found that the barnase tended to show a higher level expression in the lines whose integration sites near the distal regions of chromosomes, while the expression level became lower in the lines whose integration sites near the centromeres. This result suggested a possible relationship between chromosomal location of transgenes and the expression level. However it showed no obvious relationship between copy numbers and expression levels. In most cases, the results of multi-color FISH showed that barnase-ps1 always integrated at the same position on the chrmosome as the reporter genes(pHctinG).

[Factors influence on transformation by particle bombardment in Indica rice].

Four factors influence on transformation of indica rice, which were high osmotic treatment; different explant as the target tissue; pressure of rupture disk and quantity of plasmid DNA, were investigated in this experiment. High osmotic treatment of target tissue prior to and after bombardment increased 3.2-fold for Gus transient expression than control. The best treatment of high osmotic was that the target tissues were kept in the target-bed medium which contained 0.4-0.6 mol/L sorbitol and manitol each for 4 h prior to bombardment and for 16 h after bombardment. Four explants: scutellum from mature seed, young panicle, embryogenic callus and suspension cells of indica rice were tested as target explant by particle bombardment. The results of Gus transient showed that the highest expression was scutellum and for other three explants, the order from high to low was young panicle, embryogenic callus and suspension cell. Transgenic plants were obtained from all of the explants except young panicle. For the pressure of rupture disk on transformation, 1100 psi or 1300 psi of the pressure of rupture disk were best one for the transformation and higher than 1300 psi could damage the target tissue which become black and died in the following culture duration. For the quantity of plasmid DNA, the results showed that 0.83 microgram of plasmid DNA per bombardment was preferred for the transformation of indica rice.

[The construction of 2 BS cell lines overexpressing PKC beta I isoform and the priliminary analysis of the relationship between expression of PKC beta I and cell proliferation].

Using gene transfection technique, we have constructed a series of human embryonic lung cell lines that overexpress stably a full length cDNA encoding the beta I isoform of PKC for the first time. BS-PKC 3 is a cell line containing about 3 fold greater PKC activity than parental cell lines or control cells that carry an integrated vector lacking the cDNA inset. In comparison with control cells and parental cells, these cells exhibit significantly enhanced growth rate. The expression of oncogene c-myc which is relating closely to cell proliferation is also increased obviously in BS-PKC 3 cell. We have firstly evidence that the overexpression PKC beta I affect the level of the expression of c-myc in the 2 BS cells. It seems that there may be one of the molecular mechanisms of the effect on the proliferation in the 2 BS cells by PKC beta I.

In vitro screening of rice germplasm for resistance to brown spot disease using phytotoxin.

Two R1 IR8 plants derived from somatic cells cultured in 25% Helminthosporium oryzae toxin-medium, and one IR54 plant derived from a control (toxin free medium) were found to have mutated resistance to brown spot disease of rice. In the second generation (R2, the offspring of the R1 generation) of the three resistant populations, the segregation of resistance and susceptibility to the disease was observed. This is the first report about a disease resistant mutation obtained successfully in rice by tissue culture and in vitro screening with phytotoxin.

Somatic embryogenesis and plant regeneration in an interspecific hybrid of Oryza.

An embryogenic callus was obtained from immature panicle of an interspecific hybrid (Oryza sativa x O. latifolia) F1. The medium consisted of HE salts supplemented with 2,4-D, NAA (each 2 mg/l), kinetin (3 mg/l), yeast extract (1360 mg/l) and casein hydrolyzate (300 mg/l). The callus was milk-white in colour compact and granulate in texture. Various developmental stage of embryoid, such as globular, heart-shape, scutellum-shape and mature embryoid were observed in an embryogenic callus. Plantlets were successfully regenerated from 1-month-old callus with more than 80% regenerational frequency in each subculture for 12 passages.

Direct development of plantlets from immature panicles of rice in vitro.

Cultured immature panicles of rice formed plantlets from spikelets without callus or embryoid formation on MS and HE media containing 2 mg/l each of NAA and kinetin. Developmental stage, ploidy of explant and plant growth regulators in the medium are the major factors affecting the frequency of spikelet budding in young panicle culture. It is suggested that spikelet budding occurs by the reversion of floral primordia to vegetative stage or by the formation of adventitious buds from epidermal cells.