Inheritance of resistance toHelicoverpa armigera of 3 kinds of transgenicBt strains available in upland cotton in China

There are 3 kinds of transgenicBt strains, Shanxi 94-24, Zhongxin 94, and R19, in upland cotton in China. Their transgenicBt insect-resistance cultivars or hybrids have been developed and grown by farmers. Genetic studies indicate that the resistance of the 3 transgenicBt cotton strains toHelicoverpa armigera is controlled by one pair of non-allelic dominant genes. Linkage relationship between the resistant genes of R19 and Shanxi 94-24 transgenicBt strains shows that they may be inserted in the same chromosome. F₁ hybrids crossed among the 3 strains show that high levels of protection from feeding damage are the same as that of their parents. Therefore, there is no co-suppression phenomenon in many transgenic plants. The results presented here afford a fundamental reliance in developing transgenicBt insect-resistant cultivars and exploiting the heterosis of hybrids in upland cotton.     

Insecticidal activity of residual Bt protein at the second trophic level

Since the first commercial release of transgenic crop expressing genes from Bacillus thuringiensis (Bt), there have been concerns about its potential impact on the environment. Research has focused on the ecological effects from second exposure to Bt prot…     

Can other host species of cotton bollworm be non-Bt refuges to prolong the effectiveness of Bt-cotton?

The potential ecological risks ofBacillus thurigiensis (Bt) insecticides and Bt-crops have caused increasing concern since their commercial release in the field, among which pests’ resistance to Bt-crops is the major ecological risk. Refuge tactic, which can produce sensitive populations, has proved to be a key and sound resistance management strategy in USA and Australia; however, no tactics have been performed in China where Bt-cotton is mostly planted with other host crops of cotton bollworm. Genetic variation and gene flow among different host populations of the cotton bollwormHelicoverpa armigera were analyzed using PCR fingerprinting method. The results show that maize and castor-oil plant, as well as cotton can take effect as refuges to prevent resistance of cotton bollworm to Bt-cotton, while peanut and sesame are not as suitable for planting with Bt-cotton as refuges in the field as low gene flow was detected among populations on peanut, sesame and Bt cotton.     

Inheritance of resistance to Helicoverpa armigera of 3 kinds of transgenic Bt strains available in upland cotton in China

There are 3 kinds of transgenic Bt strains, Shanxi 94-24, Zhongxin 94, and R19, in upland cotton in China. Their transgenic Bt insect-resistance cultivars or hybrids have been developed and grown by farmers. Genetic studies indicate that the resistance of the 3 transgenic Bt cotton strains to Helicoverpa armigera is controlled by one pair of non-allelic dominant genes. Linkage relationship between the resistant genes of R19 and Shanxi 94-24 transgenic Bt strains shows that they may be inserted in the same chromosome. F₁ hybrids crossed among the 3 strains show that high levels of protection from feeding damage are the same as that of their parents. Therefore, there is no co-suppression phenomenon in many transgenic plants. The results presented here afford a fundamental reliance in developing transgenic Bt insect-resistant cultivars and exploiting the heterosis of hybrids in upland cotton.     

Effect of internal deletion of Myosin II on growth and development ofDictyostelium discoideum

Four deletion mutantDictyostelium myosin II heavy chain genes, MyΔ824-941 (Δ1/ 3S2), MyΔ934-1454 (ΔS2), MyΔ934-1194 (ΔS2-1) and MyΔ1 157–1454 (ΔS2-2), were transformed by standard electfoporation into mhcA-cells (T-null), a mutantDictyostelium cell devoid of endogenous myosin II heavy chain gene. The growth, development and formation of fruiting bodies of cells expressing those mutant myosin II s under suspension culture were investigated by comparison with the wild type cell. The results indicate that internal deletion of myosin II affeds the growth and development ofDictyastelium. Furthermore, the longer the length of deletion, the more serious the defect in phenotype.     

Observation of fiber ultrastructure of Ligon lintless mutant in upland cotton during fiber elongation

Lintless mutant is a super-short fiber mutanl in upland cotton only 4-8 mm in fiber length and also named Ligon cotton controlled by one dominant gene Li1. Fiber ultrastructure of the mutant (Li1) and its its wild (li1) in siti and in vitro was observed under an electron microscope to understand its cytological characteristics during the fiber cell elongation. The resulls showed that the mutant fiber in situ had thinner cytoplasm, more small vacuoles, less mitochondria, Golgi apparatus and endoplasmic reticula, and there were more starch granules which were free or packed in the amyloplast beside the cell wall than thai of wild type. It was indicated that scarcity of functional organelles and disability of transformation from starch to sugar might be associated with the tact that the mutant fiber cell was aborted too early to elongate into normal length. Mutant ovule in some media containing GA3 could produce a kind of huge callus that grew faster than normal ovules. The callus was covered with many white, loose, and semitransparent fiber-like cells that apt lo get off from ovule. These fiber-like cells were multicellular fibers generated by cell division and had black dots just like pigment glands in the stem and leaf of cotton. There were lots of micro-tubes beside cytoplasm membrane uf the multiecllular fiber, which were thought to be primary preparation for second wall deposition of multicellular fiber. It was indicated that GA3 might induce the expression of gene(s) that kept inactive in the field condition and then stimulate the original fiber cell in vitro to undergo divisionagain.     

Development of <Emphasis Type="Italic">Gossypium barbadense</Emphasis> chromosome segment substitution lines in the genetic standard line TM-1 of <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

Chromosome segment substitution lines （CSSL） consist of a battery of near-isogenic lines that have been developed and cover the entire genome of some crops. With the exception of one homozygous chromosome segment transferred from a donor parent, the remaining genome of each CSSL line is the same as the recipient parent. It is an ideal material for genome research and particularly QTL mapping. In the present study, we first developed one set of CSSL lines using G hirsutum acc. TM-1 （the genetic standard）, as the recipient parent and G barbadense cv. Hai7124 as the donor parent using molecular assistedlselection in BC5S1-3 generations. The CSSL consisted of 330 different lines, in which 1-4 different lines had the same or overlapping substituted segments. The genetic length of the substituted segments covered 5271.9 cM with an average segment distance of 10.9 cM, 1.5 times the total genetic length of Upland cotton （3514.6 cM）. The substituted segments of each line varied in length, ranging from 3.5 cM for the shortest segment to 23.2 cM in the longest segment. Our CSSL have not yet to cover the entire tetraploid cotton genome, due to the absence of some donor parent interval segments.     

Hybrid plant regeneration from interfamilial somatic hybridization between grapevine (vitia vinifera L.) and Red Thorowax (Bupleurum scorzonerifolium willd)

The protoplasts of Red Thorowax ( Bupleurum scorzonerifolium) irradiated by ultraviolet light (UV) at an intensity of 260μW/cm2 for 0, 1,2 and 3 min respectively were fused with that of grapevine ( Vitia vinifera). The regenerated 19 clones, every one derived from a single fused cell, were identified as hybrids by phenotype, isozyme, chromosome and 5S rDNA spacer region analysis. The results reveal that all of them are somatic hybrids. 11 hybrid calli including asymmetric and symmetric products regenerated somatic embryos and young leaves after 5 months of culture, of which 4 hybrid cell lines derived from asymmetric fusion regenerated plants with roots after 8-10 months of culture. Inspection of chromosome showed that regeneration of whole plant was related to the decrease of chromosome number. Identification of 5S rDNA spacer region of the plants confirmed that they were interfamilial hybrid plants.     

Transfer of bacterial blight resistance from <Emphasis Type="Italic">Oryza meyeriana</Emphasis> to <Emphasis Type="Italic">O. sativa</Emphasis> L. by asymmetric somatic hybridization

Asymmetric somatic hybrid plants were produced between cultivated rice (Oryza sativa L.) and wild species [O. meyeriana (Zoll. etMor, exSteud.)] with high resistance to rice bacterial blight. X-ray-irradiated protoplasts of the wild species were used as donor and chemically fused with iodoacetamide-inactivated protoplasts of rice cv. 02428 to produce hybrids. Seventy-two plants were regenerated from 623 calli based on metabolic complementation. The morphological characters of the plants closely resembled that of the rice. Simple sequence repeats were employed to identify their hybridity. Cytological analysis of root-tips revealed that their chromosome number varied in the range of 27--38. The somatic hybrids were inoculated with strains of Xanthamonas oryzae pv. oryzae at adult growth stage and demonstrated the resistance to bacterial blight introgression from the O. meyeriana.     

Expression of two insect-resistant genescryIA (b&c)/GNA in transgenic tobacco plants results in added protection against both cotton bollworm and aphids

The synthesizedBacillus thuringiensis insecticidal protein gene cryIA(b&c) and the synthesized geneGNA, (the mannose specific lectin from snowdrop (Galanthus nivalis)), tumefaciens have been inserted into plant expression vector pGW4BAI. Leave stripes ofNicotiana tabacum var. K326 have been transformed withAgrobacterium tumefaciens strain LBA4404 harboring the plant expression vector. 28 kanamycin resistant tobacco plants have been obtained. PCR and Southern blot analyses show that the foreigncryIA andGNA genes have been inserted into the genome of transformed tobacco plants. Haemagglutination assays show thatGNA has a functional activity. Leaf disc bioassays against cotton bollworm (H. armigera) show that the transgenic tobacco plants have a high insecticidal activity. The inhibition of aphid population in leaf disc bioassays againstMyzus persicae shows that the fecundity of aphid on transgenic plants is lower than that on untransformed plants; the aphid population on the transgenic tobacco plants is 25%–70% that on untransformed tobacco plants. ELISA analysis of ClyIA protein in tobcco leaves provides similar data to bioassay results. Through the two bioassays againstH. armigera andM. persicae, several transgenic tobacco plants showing high insect-resistant activities to both pests have been obtained.     

Limited ecological risk of insect-resistance transgene flow from cultivated rice to its wild ancestor based on life-cycle fitness assessment

Ecological impact caused by transgene flow from genetically engineered (GE) crops to their wild relatives is largely determined by the fitness effect brought by a transgene. To estimate such impact is critical for the ecological risk assessment prior to the commercialization of GE crops. We produced F₁ and F₂ hybrid descendants from crosses of two insect-resistant GE rice lines (Bt, Bt/CpTI) and their non-GE rice parent with a wild rice (Oryza rufipogon) population to estimate the transgenic fitness. Insect damages and life-cycle fitness of GE and non-GE crop–wild hybrid descendants as well as their wild parent were examined in a common-garden experiment. No significant differences in insect damages were observed between the wild rice parent and GE hybrid descendants under high-insect pressure. The wild parent showed significantly greater relative survival-regeneration ratios than its GE and non-GE hybrid descendants under both high- and low-insect pressure. However, more seeds were produced in GE hybrid descendants than their non-GE counterparts under high-insect pressure. Given that the introduction of Bt and Bt/CpTI transgenes did not provide greater insect resistance to crop–wild hybrid descendants than their wild parent, we predict that transgene flow from GE insect-resistant rice to wild rice populations may not cause considerable ecological risks.

     

Transgenic tobacco plants expressing synthetic Cry1Ac and Cry1Ie genes are more toxic to cotton bollworm than those containing one gene

Transgenic tobacco plants carrying CrylAc, Crylle or both genes were obtained. In the leaves of transgenic plants carrying both genes, the contents of CrylAc and Crylle proteins were 0.173% and 0.131% of the total proteins, respectively. CrylAc protein content was 0.182 % and Cry1 le protein content was 0.124% of the total proteins in the leaves of transgenic plants containing only one Bt gene. Fresh leaves of transgenic tobacco and wild-type plants were used for the insect bioassay against wild-type and Cry1Ac-resistant cotton bollworm （Helicoverpa armigera）. The bioassay results showed that transgenic plants carrying both genes were significantly more toxic to wild-type and CrylAc-resistant cotton bollworm than those carrying CrylAc or Crylle alone. This study indicates that the higher toxicity of transgenic tobacco plants carrying both genes is caused by the cooperative function of both Bt proteins, thus providing a potential way to delay the development of insect resistance to transgenic crops.     

Introduction of pokeweed antiviral protein cDNA intoBrassica napus and acquisition of transgenic plants resistant to viruses

Using cotyledonary petioles as explants, the PAP cDNA controlled by wound-inducible promoter has been introduced intoBrassica napus by coculture withAgrobacterium tumefaciens and laser microbeam puncture respectively. Regenerated plants resistant to gentamycin have been selected out. PCR amplification and Southern blotting analysis indicated that PAP cDNA together with wound-inducible promoter had been integrated intoBrassica genome with transformation frequencies of 2.0% and 1.7% for two transformation methods respectively. The test of virus challenge showed that these transgenicBrassica plants were resistant in different degrees to mechanically inoculated TuMV.     

Compound-specific carbon isotope compositions of individual long-chain n-alkanes in severe Asian dust episodes in the North China coast in 2002

Asian dust storms originating from the arid regions of central and eastern Asia and from the Loess Plateau in China frequently occur in spring [1,2]. Driven by the East Asian monsoon, Asian dust can be transported to northern Pacific Ocean[2―8]. Asian du…     

Inheritance of the delayed gland morphogenesis trait in Australian wild species ofGossypium

Five Australian wild cotton species with the delayed gland morphogenesis trait, as well as G. arboreum, G. davidsonii and four different gland genotypes of G. hirsutum, Gl₂Gl₂Gl₃Gl₃, Gl₂Gl₂gl₃gl₃, gl₂gl₂Gl₃Gl₃, and gl₂gl₂gl₃gl₃, were used in this experiment and 10 interspecific hybrids were obtained by the crossing among them. According to the gland expression on the seeds and plants of the interspecific hybrids, the inheritance of the delayed gland morphogenesis trait of Australian wild cotton species was opened out as follows: (ⅰ) the inheritance of the delayed gland morphogenesis trait was almost the same among the 5 Australian wild cotton species, and the gene or genes which controlled this trait may be located in the same loci. (ⅱ) The glandless seed trait of the Australian wild cotton species was dominant over the glanded seed trait of G. arboreum, a genome A species, and the seeds of interspecific hybrid F₁ between them were glandless. However, it was recessive over the glanded character of genome D species, G.davidsonii, and their F₁ was a typical glanded one. (ⅲ) The glandless seed trait of the Australian wild cotton species was recessive or incomplete dominant over the glanded cotton but dominant over the glandless cotton of G. hirsutum, and the glandless genes (gl₂gl₂gl₃gl₃) of upland cotton had great weakening effect on the glanded plant trait of the Australian wild cotton species on the other hand. For the two main glanded genes of upland cotton, the delayed gland morphogenesis trait of the Australian wild cotton species was dominant epistatic over glandless genes, gl₂gl₂gl₃gl₃, and one of the glanded genes, Gl₂Gl₂, but was recessive epistatic over the other glanded gene, Gl₃Gl₃. Therefore, it is much convenient to use Gl₂Gl₂gl₃gl₃ as the upland cotton parent in the interspecific hybridization and backcrossing afterward, in order to produce the upland cotton germplasm with glandless seeds and glanded plant trait.     

Introduction of antisensewaxy gene into main parent lines ofindica hybrid rice

Amylose content in rice endosperm is one of the key determinants of rice eating and cooking quality, and the poor quality ofindica hybrid rice is closely related to the high amylose level in rice grains. In order to improve the grain quality of theindica hybrid rice by genetic engineering, an antisense fragment of ricewaxy gene, driven by the 5′-franking sequences of the ricewaxy gene, was successfully introduced into three major parent lines ofindica hybrid rice, all contain a high amylose level in the grains, viaAgrobacterium, and more than 100 hygromycinresistant plants were regenerated. The analysis of PCR amplification and Southern blots indicated that the T-DNA containing the antisensewaxy gene had been integrated into the genome of transgenic rice plants. Most of the primary transgenic rice plants grew normally, and the mature seeds from these transgenic plants were performed for analysis of the amylose content. The results showed that the amylose content in the endosperm of some grains was reduced and the lowest reached 7.02% in one homozygous transgenic line, 72.4% lower than that of the wild type. The influence of the altered amylose content on the gelatinization temperature and gel consistency was also observed in several homozygous transgenic rice plants. The two authors contributed equally to this work.     

Genetic analysis and gene mapping of a narrow leaf mutant in rice ( Oryza sativa L.)

A narrow leaf mutant was obtained after T-DNA transformation conducted on a rice variety Zhonghua 11. Several abnormal morphological characteristics, including semi-dwarf, delayed flowering time, narrow and inward rolling leaves, and lower seed-setting, were observed. The rate of net photosynthesis (under saturate light) of flag leaves in the mutant was significantly lower than that of the wild type. Moreover, the leaf transpiration rate and stomatal conductance in the mutant flag leaf were lower than those of the wild type at the grain filling stage. It was found that the mutant phenotype was not caused by the T-DNA insertion. Genetic analysis showed that the mutant was controlled by a single recessive gene, designated as nal3(t). A genetic linkage map was constructed using a large F₂ mapping population derived from a cross between nal3(t) and an indica variety Longtefu B with 6 polymorphic markers on chromosome 12 identified from 366 SSR markers by the BAS method. Gene nal3(t) was mapped between the markers RM7018 and RM3331. Fine mapping of nal3(t) locus was conducted with 22 newly developed STS markers based on the sequence diversity around the region harboring nal3(t) between Nipponbare and 93–11, and nal3(t) was finally mapped to a 136-kb region between the STS markers NS10 and RH12-8. Supported by National High Technology Research and Development Program of China (863 Program) (Grant No. 2006AA10A102), National Natural Science Foundation of China (Grant No. 30600349) and Natural Science Foundation of Zhejiang Province (Grant No. Y306149)     

Genetic analysis and molecular mapping of a presenescing leaf gene psll in rice （Oryza sativa L.）

A rice psl1 （presenescing leaf） mutant was obtained from a japonica variety Zhonghua 11 via radiation of ^60Co-γ in M2 generation. Every leaf of the mutant began to wither after it reached the biggest length, while the leaves of the wild variety could keep green for 25--35 d. In this study, genetic analysis and gene mapping were carried out for the mutant identified. The SSR marker analysis showed that the mutant was controlled by a single recessive gene （psl1） located on chromosome 2. Fine mapping of the psl1 locus was conducted with 34 new STS markers developed around psl1 anchored region based on the sequence diversity between Nipponbare and 93-11. The psl1 was further mapped between two STS markers, STS2-19 and STS2-26, with genetic distances of 0.43 and 0.11 cM, respectively, while cosegregated with STS2-25. A BAC contig was found to span the psl1 locus, the region being delimited to 48 kb. This result was very useful for cloning of the psl1 gene.     

Agrobacterium-mediated transformation: state of the art and future prospect

Great progress has been made in recent years in studies on the mechanism ofAgrobacterium-medicated 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 ofAgrobacterium, can now be transformed by this bacterium, and they include the very important cereal species, gymnosperms, yeast and many filamentous fungi. The simplein 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 byAgrobacterium. Further studies will elucidate more plant-encoded factors involved in T-DNA transformation and there is a need to develop more powerfulAgrobacterium-based transformation systems to meet different needs in basic research and crop improvement practice.