Great progress has been made in recent years in studies on the mechanism of Agrobacterium-mediated transformation and its application. Many details of the key molecular events within the bacterial cells involved in T-DNA transfer have been elucidated, and it is notable that some plant factors which were elusive before are purified and characterized. Vast kinds of species, which were either recalcitrant to or not included in the host range of Agrobacterium, can now be transformed by this bacterium, and they include the very important cereal species, gymnosperms, yeast and many filamentous fungi. The simple in vivo transformation of tissue in intact plants and the "agrolistic" methods to transform recalcitrant plants are the two novel technical achievements. Combined with other powerful techniques such as bacterial artificial chromosome, very large DNA fragment can be transformed into the plant genome by Agrobacterium. Further studies will elucidate more plant-encoded factors involved in T-DNA transformation and there is a need to develop more powerful Agrobacterium-based transformation systems to meet different needs in basic research and crop improvement practice.

A method for fast plant regeneration via organogenesis directly from Lycium barbarum leaf explants has been developed. The key factor for shoot regeneration was the presence of benzyladenine (BA) in the medium. NAA could only induce root formation and explant callusing. Murashige and Skoog (MS) medium supplemented with 2mg/l BA and 0.5mg/l NAA is the most efficient condition for shoot formation, with up to 92.6% shoot regeneration and no callus formation. All adventitious shoots cultured on MS medium supplemented with 1mg/l IAA formed an extensive root system. Regenerated plants were morphologically normal and were also proved to be diploid (2n=24). Using the optimized regeneration system, the genetic transformation of L. barbarum was carried out mediated by Agrobacterium tumefaciens EHA101(pIG121Hm). 11.8% leaf explants produced kanamycin-resistant shoots after infection by A. tumefaciens. The putative transgenic nature of plants was confirmed by GUS assay and PCR analysis. Expression of the nptⅡ gene in the regenerated plants was also detected by observing the callus formation by leaf pieces on MS medium containing 0.2mg/l 2,4-D and 0-100mg/l kanamycin.

We have developed a reliable and high-frequency system of transformation and regeneration via somatic embryogenesis (SE) of Lycium barbarum. Leaf segments were co-cultivated with Agrobacterium tumefaciens EHA101 (pIG121Hm) carrying the neomycin phosphotransferase II gene as a selectable marker and an intron-β-glucuronidase (GUS) gene as a reporter marker. On the medium for callus-induction, which contained 50mg l-1 kanamycin (Km), approximately 60% of the explants produced kanamycin-resistant (KmR) calli. After three subcultures on the same selective medium, KmR calli were transferred onto SE induction medium containing 25mg l-1Km, where they produced numerous somatic embryos (166 g-1fresh weight callus) that subsequently developed into KmR plantlets. Compared with the untransformed control calli, the transformed calli maintained their higher frequency of SE up to 16 months after transformation. GUS staining and polymerase chain reaction and Southern blot analyses confirmed the integration of the T-DNA into the plant genome.

An effective protocol has been developed for plant regeneration from cotyledon explants of Swainsona salsula Taubert (Saline swainsona), a medicinal and agronomic shrub. Adventitious shoots were obtained from 83.2% of cotyledon explants from 3-day seedlings cultured on Murashige and Skoog (MS) medium containing 2.0mgl−1 thidiazuron (TDZ), with an average of 9.3 shoots per explant. Individual elongated shoots were rooted on half strengthMS medium supplemented with 2.0mgl−1 indole-3-butyric acid (IBA), with 59.3% success. Regenerated plants with well developed shoots and roots were successfully transferred to soil, without detectable variants. Histological observation revealed that shoots developed from cotyledon explants via organogenesis, with little callus.

In oogamous reproduction of multicellular organisms, a striking phenomenon is the prevailing synchronous development of male germ cells connected by wide intercellular bridges (IBs, 0.1-2mm), which is well conservedin both animal andplant species ranging from algae to human. In the literature, IBs are believed either to allow genetically segregated haploid spermatids to share diploid gene products after meiosis, or to mediate rapid transfer of some vital signals or nutrients. Although intercellular sharing of gene transcripts has experimental evidences, these hypotheses are still not satisfactory. To explore the unknown roles of IB, we assume that developing male germ cells may be especially sensitive to stochastic gene expression to become heterogeneous. To achieve best gamete quality, such heterogeneity must be eliminated so that relatively uniform gametes with normal functions can be produced. Development within a common syncytium may be the only way for this purpose. The process may require not only the intercellular exchange of a few molecular signals but also the mixing of protoplasm between the connectedcells so that they have similar levels/states of mRNAs, proteins andorganelles, which can be achievedonly through wide IBs. This hypothesis can explain some quite intriguing aspects of male gametogenesis and provide unique predictions that can be tested experimentally.

Development of an efficient in vitro plantlet regeneration system from tubers of Stachys sieboldii (Miq.), a traditional Chinese medicinal plant and vegetable, is described. Adventitious shoots with an average of 9.1 (after 4 weeks of incubation) shoots per explant were obtained from 60.4% of tuber explants cultured on Murashige and Skoog (MS) medium containing 1.0mgl−1 thidiazuron (TDZ), and after another 4 weeks of incubation on growth regulator free MS medium, 33.5 shoots per explant were achieved. Subcultures showed results that were similar to those of the initial culture. Excised shoots were rooted both on MS and on half strength MS medium with a frequency of about 95%. Excised shoots were also rooted ex vitro by directly planting them into cups containing sand. Regenerated plants with well developed shoots and roots were successfully transplanted to soil, after which they developed free of abnormalities.

A micro-propagating system based on the young stem node segments of Sophora flavescens Ait. (Fabaceae) was established. Murashige and Skoog (MS) basal medium supplemented with 8.88μM 6-benzyladenine (BA) plus 2.69μM a-naphthalene acetic acid (NAA) and that with only 5.37μM NAA were found the best in promoting proliferation of shoots and induction of root, respectively. Percentages of shoot induction and number of shoot per explant were up to 93.4% and 4.2 and rooting rate to 82.4%, respectively. The segments of the regenerated shoots could be continuously induced to reproduce new shoots through subculture on the same medium in 30-d intervals and still kept this activity after being subcultured for 6 generations. After the regenerated plantlets were transplanted to field, they grew well, showing no any visible abnormalities.

Using apical and axillary nodes as explants, a rapid and efficient method for propagation of Amorphafruticosa L. has been developed. When grown on Murashige and Skoog (MS) medium supplemented with 8mg dm-3 benzyladenine, 100% explants responded with 4.94 shoots per explant after 6-weeks culture, and explants taken from the in vitro proliferated shoots subsequently produced multiple shoots when cultured on the same medium. The addition of casein hydrolysate (200mg dm"3) enhanced the number of shoots up to 8.77 per subculture, and coconut milk was found to promote the shoot elongation and make them grow more vigorously. 82.53% excised shoots were rooted on half-strength MS medium containing 2.0mg dm"3 indoleacetic acid after 3 weeks of incubation. After acclimatization, all of the rooted plantlets established in soil, exhibiting uniform morphological and growth characteristics.