Rapid and convenient transformation of cotton (Gossypium hirsutum L.) using in planta shoot apex via glyphosate selection

Cotton plants are recalcitrant with regards to transformation and induced regeneration.In the present study,5-enolpyruvylshikimate-3-phosphate（EPSPS）,a glyphosate resistant gene from the bacterium Agrobacterium sp.strain CP4,was introduced into an elite Bt transgenic cotton cultivar with a modified technique involving in planta Agrobacteriummediated transformation of shoot apex.Primary transformants were initially screened using a 0.26%glyphosate spray and subsequently by PCR analysis.Five out of 4 000 transformants from T_1 seeds were obtained resulting in an in planta transformation rate of 0.125%.Four homozygous lines were produced by continuous self-fertilization and both PCR-based selection and glyphosate resistance.Transgene insertion was analyzed by Southern blot analysis.Gene transcription and protein expression levels in the transgenic cotton lines were further investigated by RT-PCR,Western blot,and ELISA methods.Transgenic T_3 plants were resistant to as much as 0.4% of glyphosate treatments in field trials.Our results indicate that the cotton shoot apex transformation technique which is both tissue-culture and genotype-independent would enable the exploitation of transgene technology in different cotton cultivars.Since this method does not require sterile conditions,the use of specialized growth media or the application of plant hormones,it can be conducted under the greenhouse condition.

Science Letters:Optimization of soybean (Glycine max (L.) Merrill) in planta ovary transformation using a linear minimal gus gene cassette

Soybean transformation by ovary-drip was improved by optimizing the length of the transformation pathway by cutting the styles. These modifications facilitated soybean transformation manipulation and improved transformation reproducibility and efficiency. Using a linear minimal gus gene cassette as the foreign DNA, a maximum transformation frequency of 11% was obtained in flowers of the soybean cultivar ‘Liaodou 14’ with their styles mostly removed, whereas removal of only the stigma, partial style cutting and partial ovary cutting gave transformation frequencies of 0%, 1%, and 2%, respectively. An average transformation frequency of 8.2% was obtained when 619 flowers from three soybean cultivars (‘Liaodou 14’, ‘Liaodou 13’, and ‘Tiefeng 29’) were transformed by this optimized method. Southern blotting analysis showed that the gus reporter gene (encoding β-glucuronidase) was stably inherited with a simple pattern. Reverse transcription-polymerase chain reaction (RT-PCR) and GUS staining confirmed the expression of the gus gene in transgenic plants.

Transformation of Chinese Cabbage(Brassica rapa L. ssp. pekinensis)by Agrobacterium Micro-Injection into Flower Bud

We obtained two lines of Chinese head cabbage(Brassica rapa L. ssp. pekinensis)selfed progenies containing both an anti-sense gene of BcpLH and a gene for resistance to kanamycin by micro-injecting buds of their primary transformants(T0)with Agrobacterium tumefaciens strain LBA4404. 31 positive plants resistant to kanamycien were recovered. Southern blot analysis confirmed the presence of T-DNA in two transgenic plants. One(DHZ-13-1)exhibits the characteristics of out-toward rosette and cauline leaves, and nested flower model in which secondary complete flower developed from the base of the primary ovary and the third flower from the ovary in the secondary flower, and so on, while another(DHZ-6-1)has no phenotype change. ABA and IAA affected the root growth of progeny of DHZ-13-1, but 6-BA was insensitive to hypocotyl growth during its seedling development.

Plant as Bioreactor for Producing Heterologous Proteins

Using plants to produce heterologous proteins makes it very attractive due to the potentially low costs. Using this procedure it is possible to produce medicinal protein for clinical applications with the plants bioreactors increasing gradually. The paper proposes the five major systems of the plant bioreactor as well as their advantage and disadvantage and the development of each system. Focuses on the five major systems of the plant bioreactor to produce vaccines, antibodies and medical protein and the research achievement at the present stage and the research on my laboratory. The key technology research of plant bioreactor such as new genes, new biological components, new technologies and new research methods related with plant bioreactor offer a work foundation for a long-term development in future.

水稻白叶枯病菌在寄主体内外不同生长条件下致病基因差异表达的分析

To demonstrate the expression profiling of Xanthomonas oryzae pv.oryzae(Xoo) in vitro and in planta,DNA microarrays of 371 genes potentially associated with pathogenicity and virulence were used to compare the transcriptional level alteration of the wild-type strain PXO99A and gene deletion mutants ΔgacAxoo and ΔfleQxoo of Xoo grown in the rich medium NBY vs.hrp-inducing minimal medium XOM2 or leaf tissues of rice.Results indicated that 17 and 38 genes of PXO99A were differentially expressed in XOM2 and the leaf tissues of rice relative to NBY,respectively.Twenty-eight genes of ΔgacAxoo grown in XOM2 and 12 genes of ΔfleQxoo in NBY were differentially expressed relative to PXO99A.The identification of differentially-expressed genes,GacAxoo-and FleQxoo-regulons and novel candidate genes of Xoo strains would provide us the target genes for further functional analysis in pathogenesis of Xoo.