Developments and prospects for doubled haploid wheat

Doubled haploid production is a valuable biotechnology that can accelerate the breeding of new wheat varieties by several years through the one-step creation of 100% homozygous plants. The technology also plays important role in studying the genetic control of traits in wheat, in marker-assisted selection, in genomics and in genetic engineering. In this paper, recent advances in androgenesis and gynogenesis techniques, emphasizing predominantly the in vitro culture phase, as well as the emerging innovative approaches in researching and producing wheat doubled haploids are reviewed. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- based genome editing, that allows targeted mutagenesis and gene targeting, is being tested extensively as a powerful and precise tool to induce doubled haploids in wheat. The review provides the reader with recent examples of gene modifications in wheat to induce haploidy.Serik Eliby, Sara Bekkuzhina, Olena Kishchenko, Gulnur Iskakova, Gulnar Kylyshbayeva, Satyvaldy Jatayev, Kathleen Soole, Peter Langridge, Nikolai Borisjuk, Yuri Shavruko

Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation

Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.Проанализирована способность lox-сайтов Cre/lox системы рекомбинации бактериофага Р1 влиять на экспрессию трансгенов при расположении этой последовательности непосредственно возле правого бордера (RB) перед кодирующей последовательностью гена. Нативная и мутированная последовательность lox-сайта были размещены в векторах для трансформации возле гена bar и проведена генетическая трансформация растений с помощью агробактерии и биолистическим методом. Lox-опосредованная экспрессия гена bar, обусловливающая устойчивость растений к фосфинотрицину, наблюдалась только у растений, которые получены с помощью агробактериальной трансформации. Методом РТ-ПЦР анализа подтверждено, что в трансгенных растениях, устойчивых к фосфинотрицину, происходит транскрипция гена bar. Сконструирован вектор, в котором ген gus и предшествующий ему lox-сайт размещены вблизи правого бордера, и проведена трансформация табака этим вектором. Экспрессия гена gus задетектирована в листьях трансгенных растений. Векторы, у которых последовательность lox-сайта предшествует гену bar возле правого бордера (RB-lox-bar), успешно использованы для получения устойчивых к фосфинотрицину трансгенных растений таких видов, как Beta vulgaris, Brassica napus, Lactuca sativa и Solanum tuberosum. Наши результаты подтверждают возможность использования последовательности lox-сайта возле правого бордера для контроля экспрессии гена bar в трансгенных растениях

A method for the generation of combinatorial antibody libraries using pIX phage display

For more than a decade, phage displayed combinatorial antibody libraries have been used to generate and select a wide variety of antibodies. We previously reported that the phage coat proteins pVII and pIX could be used to display the heterodimeric structure of the antibody Fv region. Herein, aspects of this technology were invoked and extended to construct a large, human single-chain Fv (scFv) library of 4.5 × 10(9) members displayed on pIX of filamentous bacteriophage. Furthermore, the diversity, quality, and utility of the library were demonstrated by the selection of scFv clones against six different protein antigens. Notably, more than 90% of the selected clones showed positive binding for their respective antigens after as few as three rounds of panning. Analyzed scFvs were also found to be of high affinity. For example, kinetic analysis (BIAcore) revealed that scFvs against staphylococcal enterotoxin B and cholera toxin B subunit had a nanomolar and subnanomolar dissociation constant, respectively, affording affinities comparable to, or exceeding that, of mAbs obtained from immunization. High specificity was also attained, not only between very distinct proteins, but also in the case of the Ricinus communis (“ricin”) agglutinins (RCA(60) and RCA(120)), despite >80% sequence homology between the two. The results suggested that the performance of pIX-display libraries can potentially exceed that of the pIII-display format and make it ideally suited for panning a wide variety of target antigens