[Construction of a novel vector harboring green fluorescence protein gene (gfp) and high expression of gfp in transformed roots of Petunia hybrida].

A novel vector pBIN-35S-GFP was constructed from the plasmids of pBIN19, pGFP, and pCHS, which included gfp gene driven by the CaMV 35S promoter. The hairy roots of Petunia hybrida were induced by wild-type Agrobacterium rhizogenes K599 harboring pBIN-35S-GFP with the frequency of 45%. The PCR results showed that rolB from K599 Ri plasmid and gfp from pBIN-35S-GFP were co-transformed into the genome of P. hybrida. The high activity of green fluo-rescence protein was detected by fluorescence microscopy. In particularly, the vector carries multiple cloning sites at both 5' and 3' of the CaMV 35S promoter, which allows easy exchange 35S promoter to study other promoter functions. In addition, there are multiple cloning sites at 5' end and one-sites of EcoRand Bsmsites at 3' end of gfp. Therefore, it supports to fusion target genes to expression fusion protein and can be replaced with any other genes of interest for genetic transforma-tion.

[Studies on the megasporogenesis and microsporegenesis and the development of the female gametophyte and male gametophyte in Camptotheca acuminate].

The ovarium is hypostasy in Camptotheca acuminate Decne.. It has a locule and an ovule. The ovule is pendulous, anatropous andunitegmic. The ovule of Camptotheca acuminate Decne. is pseudocrassinucellate ovule. The development of embryo sac is polygonum type. Cytokinesis during the meiosis of microspore mother cells is of simultaneous type. The arrangement of microspores in tetrad is tetrahedral and isobilateral. One-nucleate microspore is triangle. Maturity pollen is triangle, circular and square. This paper mainly studied the megasporogenesis and microsporogenesis, and studied the development of their female and male gametophyte in Camptotheca acuminate Decne., and preliminarily discussed the cause of the part pistil abortion in Camptotheca acuminate Decne.

Sequence analysis of a novel insertion site of transposon IS10.

A sacB mutant was obtained by transposon IS10 inactivation of a plasmid pXT3sacB carrying the sacB gene. Sequencing of this mutant plasmid DNA (GenBank accession No. AY580883.1) showed that the IS10 flanking the 22 bp inverted repeats were 5'-CTGAGAGATCCCCTCATAATTT-3' and 5'-AAATCATTAGGGGATTCATCAG-3', which were the similar to those published in reports previously. However, the target sequence adjacent to IS10 was 5'-TGCTTGGTT-3' instead of the previously reported 5'-NGCTNAGCN-3'. To our knowledge, this is the first report on the novel insertion site of IS10. In addition, Southern blot hybridization confirmed that the mobile IS10 originated from the chromosomal DNA of the host strain Escherichia coli DH5alpha and that there were two copies in the DH5alpha genome.

[Morphological studies on direct regeneration of floral buds from in vitro cultures of sepal segments in Sinningia speciosa hiern].

The morphological changes in the cultures of sepal segments in Sinningia speciosa Hiern were observed with Zeiss Stemi 2000-C Stereomicroscope from 0 to 65 days after culture in vitro. The light yellow globular protuberances were observed on the cut edge and the surface of sepal segments after culture for 24 days. Then the globular protuberances grew bigger gradually. A lot of floral buds on the surface of sepal segments formed after culture for 60 days. The morphological changes in the cultures of sepal segments were studied with light microscopy after culture for 0 to 30 days as well. The cells of tissues of sepal segments arranged regularly and no dividing cell was observed on the first day culture. There appeared some small meristematic centers of dividing cells on cut edge and the lower epidermis on the 7th day after culture. To the 20th day culture in vitro, the floral organ primordia were differentiated on the cut edge. On the 30th day culture, floral buds with petal, stamen primordia were observed. In addition, the morphological changes in the cultures of sepal segment were studied during the 14 to 30 days culture with scanning microscopy as well.

[Hairy root induced by wild-type agrobacterium rhizogenes K599 in soybean, cucumber and garden balsam in vivo].

The plantlets of soybean, cucumber and garden balsam were inoculated by wild-type Agrobacterium rhizogenes K599, and hairy root was induced on inoculated sites in vivo. The frequencies of hairy root induction from wound cotyledons of soybean, cucumber and garden balsam were 100%, 65% and 91%, respectively. Moreover, hairy root was induced from healthy cucumber axillary bud with frequency of 10%. PCR analysis of hairy root DNA was conducted using the primers from rolC gene. The PCR results showed that all hairy root lines contained T-DNA. The established system should be ideal for studying soybean and cucumber nematode and garden balsam breeding of flower dwarf architecture.

[Studies on the megasporogenesis and microsporogenesis and the development of their female and male gametophyte in Magnolia biloba].

The anther of Magnolia biloba is tetrasporangiate with glandular tapetum, which consists of one or two layers of cells. Cytokinesis during meiosis of its microspore mother cell is modified simultaneous type, and the microspore tetrads are isobilateral. Mature pollen grains are two-celled. Tetrad cells and microspores are irregularly shaped during the microsporogenesis. There were two ovules on the ventral surface of unicarpellate ovary wall. Ovules were anatropous, bitegmnous and crassinucellar. Archesporial cell was one cell and differentiated from cell in the second layer beneath epidermis. The development of the embryo sac conformed to the polygonum type. The embryological characteristics of Magnolia biloba are very similar to those of other species in Magnoliaceae. The megasporogenesis and microsporogenesis and the development of their female and male gametophyte are partially abnormal. Abnormal phenomena in the process of reproduction of Magnolia biloba causing this species to be endangered was discussed.

[Cloning and sequencing of fragments associated with cytoplasm male sterility of rice].

Mitochorndrial DNA (mtDNA), nuclear DNA (nDNA), and genomic DNA (gDNA) were individually extracted from Zhenshan 97A, Xieqingzao A and A-23, which were cytoplasmic male sterile (wild abortive, dwarf abortive and BT), and from Zhenshan 97B, Xieqingzao B and BT, which were maintainers lines. Each of them was analyzed with 500 random primers by RAPD. Six fragments specific to male sterile lines were amplified. A fragment, PWH-17, associated with wild and dwarf abortive type cytoplasmic male sterility was analyzed by Southern blotting and sequencing, and also tested by SCAR. It was 1879 bp in length, which contained 6 open reading frames and 8 repeated sequences. BLAST search revealed that partial sequence in PWH-17 showed high similarity to the upstream sequence of tRNA-Asp gene in Elytrigia elongate (GenBank accession number U14335) and wheat (X13243). The similarity ratios were 97% and 100%. It was inferred that the possible position of PWH-17 in rice (wild abortive) could be located between tRNA-Asp and Cox II, which will be discussed further.

[Lyophilized mixture of PCR ingredients and its application].

In this paper we introduce a new storage method of PCR ingredient. PCR mixture except DNA template has been frozen to dry powder by the DNA-Plus system. This kind of powder was stored at room temperature or 4 degrees. PCR has been run in different period of storage. It was discovered that the samples of lyophilization could keep activity for a long time.

[A template DNA preparation method for PCR and RAPD reaction by using alkali-treated wheat leaf tissues].

In this paper we introduce a new method of PCR and RAPD reaction by using a small quantity of wheat leaf tissue with alkali treatment. It can be used either to detect target gene or to analyze polymorphism of wheat varieties. This method is simple and reliable, especially for a large-scale detection.