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Calcium-dependent protein kinases play important roles in the regulation of plant growth and development. Rice genome contains 31 CDPK genes, and the biological functions of most of these CDPK genes remain unclear. To elucidate the biological function of OsCPK25/26, we first analyzed the expression profiles for OsCPK25/26. Transcripts of OsCPK25/26 were observed in the panicles, hulls, and stamens, with significantly higher expression in stamens. Plasmid constructs of either overexpression of OsCPK25 or RNAi knockdown of OsCPK25/26 were created and introduced into the Zhonghua11 (Oryza sativa L. ssp. japonica) rice variety, and single-copy homozygous lines of the resultant transgenic plants were characterized. The expression levels of OsCPK25/26 in panicles were increased 11–15-fold in the overexpressing plants and decreased 1.5–1.7-fold in the plants expressing RNAi against OsCPK25/26 compared with that in the control plants. The numbers of stamens in some florets increased with varying degrees, ranging from six to 13 stamens in both the overexpressing and RNAi plants. Compared with the control plants, the proportion of florets with increased stamen numbers in T2 generation was 30 % in the overexpressing plants and 36 % in RNAi plants and that in T3 generation was 50 and 51 %, respectively. Our results suggest that OsCPK25/26 are involved in the regulation of stamen development, and changes (increases or decreases) of OsCPK25/26 expression levels result in an increased number of stamens. Thus, the optimal expression levels of OsCPK25/26 are a key regulating factor in maintaining normal stamen numbers in rice.

In recent years, transgenic pyramiding or stacking technology has gradually developed with the rapid development of genetically modified (GM) crops. This technology has unique advantages compared with the transgenic technology of a single gene. Transgenic pyramiding technology can simultaneously modify several traits of a crop, particularly the metabolic pathways and yield traits that are usually controlled by multiple genes. A batch of second generation GM crops with stacked traits has been commercialized, which mainly include resistances to pest and herbicide, and exhibit great application potential of transgenic pyramiding breeding. The present stacked GM crops are developed mainly through the integrated use of vector-based pyramiding and molecular marker assisted selection (MAS) based crossing/backcrossing pyramiding. Advances of vector based pyramiding technologies have been achieved on large capacity vectors, multiple-gene assembling, plastid transformation, polyprotein expression system and combinatorial genetic transformation. The combination of transgenic pyramiding and MAS-based pyramiding will help to effectively pyramid more targeted genes together. The advance of single nucleotide polymorphism (SNP) discovery and detecting technology has greatly promoted the MAS-based pyramiding in crop breeding. The rapid development of commercialized stacked GM crops, plant metabolic engineering and crop improvement for disease resistance have proved the successful applications of transgenic pyramiding. The present review discusses the advances of transgenic pyramiding technology, the application of transgenic pyramiding in crop improvement and the prospects and challenges of transgenic pyramiding breeding.

Tobacco leaf discs were transformed with a plasmid, pBIAHA, containing the selectable marker neomycin phosphotransferase gene (nptII) and an Arisaema heterophyllum agglutinin gene (aha) via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic tobacco plants were regenerated. PCR and Southern blot analyses confirmed that multiple copies of the aha gene had integrated into the plant genome. Northern blot analysis revealed that the aha gene was expressed at various levels in the transgenic plants. Insect bioassay test showed that transgenic plants expressing multiple copies of the aha gene reduced the rate of population increase of the peach potato aphid (Myzus persicae Sulzer). This is the first report that transgenic tobacco plants expressing the aha gene display enhanced resistance to aphids. Key words: Insect bioassay, Arisaema heterophyllum agglutinin, transformation, transgenic tobacco, peach potato aphid (Myzus persicae Sulzer)

Mature seed-derived callus from an elite Chinese japonica rice cv. Ewan 5 was cotransformed with two plasmids, pWRG1515 and pRSSGNAl, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis) lectin gene (gna) via particle bombardment. Thirty-five independent transgenic rice plants were regenerated from 177 bombarded calluses. Eighty-three percent of the transgenic plants contained all three genes, as revealed by Southern blot analysis. Western blot analysis revealed that 23 out of 29 gna-containing transgenic plants expressed Galanthus nivalis agglutinin (GNA) (79%) at various levels, with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of all three transgenes (gna, hpt and gusA) in the R2 progeny. Amongst the R2 generation two independent homozygous lines were identified that expressed all three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper (Nilaparvata lugens, BPH) by decreasing the survival, overall fecundity of BPH, retarding development, and decreasing the feeding of BPH. These BPH-resistant lines have been incorporated into a rice insect resistance breeding program. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH.

Mature seed-derived callus from an elite Chinese japonica rice (Oryza sativa L.) cv. Eyi 105 was co-transformed with two plasmids, pWRG1515 and pRSSGNAI, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snow-drop (Galanthus nivalis) lectin gene (gna) via particle bombardment. After two rounds of selection on hygromycin-containing medium, resistant callus was transferred to hygromycin-containing regeneration medium for plant regeneration. Twenty-six independent transgenic rice plants were regenerated from 152 bombarded calli with a transformation frequency of 17%. Seventy-three percent of transgenic plants contained all three genes, which was revealed by PCR/Southern blot analysis Thirteen out of 19 transgenic plants containing the gna gene expressed GNA (68%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parent plants showing 3:1 Mendelian segregation patterns, we identified three independent homozygous lines containing and expressing all three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to the rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and reducing BPH feeding. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.