Sequences of two recently cloned genes playing a role in resistance against wheat pathogens (receptor-like kinase Lrk10 and Cre3 genes) were used to search for similarity of cereal clones included in the NCBI database. We found 23 clones with similarity to the Cre3 gene with predicted NBS and LRR domains, and 50 clones with serine/threonine kinase function and similarity to the new receptor-like kinase gene Lrk10 from wheat. In those two groups of clones some conservative nucleotide sequences were identified. Two sequences are identical between the majority of resistance gene candidate clones with a high similarity to Lrk10, and two sequences are identical between the majority of resistance gene candidate clones with similarity to the Cre3 gene.
This paper provides an overview of the development of methods of cereals doubled haploid production over the last decade. The influence of genotype and albinism remain the main problem of androgenesis. Localisation of major genes influencing androgenic potential and manipulation with temperature during induction and regeneration offer possibilities of efficiency improvement. Isolated microspore culture in cereals is effective when microspores are co-cultured with sporophytic tissue. Gynogenic methods may be developed for some barley and apomictic wheat forms. Distant crosses that are followed by the elimination of chromosomes of the male parent from hybrid embryos are broadly applied. Maize and its relatives are used as effective pollinators of a wide range of cereal species since prezygotic barriers have not been found so far. Brief description of utilisation of doubled haploid lines in breeding programs, research and genetic transformation of cereals closes the overview.
The development of microspore culture methods in the Poaceae family has received considerable attention in recent years. Isolated microspore culture can be induced in vitro to switch their development from gametophytic to a sporophytic patway, resulting in embryoid or callus formation. Different stresses like cold or heat shock and nitrogen starvation have been identified as the major trigger in inducing microspore embryogenesis. Microspore culture appears to be a promising toll for future production of double haploids in cereals. Isolated microspore culture has several advantages over anther culture in genetic manipulation and haploid study, such as: direct observation of microspore development, unique possibility to study plant embryogenesis, easier in vitro selection and mutation, easier transformation of single cells. Moreover, isolated microspores are the most efficient way of double haploid regeneration. Many factors such as genotypes, physiological status of donor plants, stage of microspores development, pretreatment of anthers or spikes, method of microspores isolation, culture media, nurse culture and culture conditions, have a great influence on microspore culture. These and other problems concerning in vitro culture of isolated microspore are discussed in this review.
RNAi technology is based on a natural process of RNA-directed gene regulation. The technique is widely used for gene functional analysis and to obtain plants with modified traits. The main advantage of this system, particularly when applied for polyploid species, is the possibility of simultaneous silencing of homologous, homoeologous or orthologous genes. The article discusses the results of relatively few papers where RNAi has been used for functional analysis of native genes of wheat and barley. The main part of the article presents the research on RNAi based gene silencing in cereals performed by our group. The experimental basis of our work was the elaboration of efficient Agrobacterium-based transformation and plant regeneration systems of different cereal species (wheat, barley, triticale and oat). Currently, the method is applied for modification of two types of traits in wheat, triticale and barley. The first one is a technological trait related to cereal grain hardness. It is genetically controlled by Pina and Pinb genes. We obtained over a hundred transgenic lines with various degrees of Pina and Pinb silencing. Currently, the lines are being analyzed for the amount of PINA and PINB proteins, composition of storage proteins, and the grain texture. The second set of traits depends on CKX genes encoding cytokinin oxidase/dehydrogenase ? the part of the system specifically governing the cytokinin level in different organs and developmental stages. We obtained over forty barley transgenic lines with silenced HvCKX1. This modification was found to be tightly correlated with enhanced plant productivity measured as the higher grain number and higher mass of a thousand kernels. The T1 and T2 transgenic seedlings developed bigger root system.
The paper reviews somatic hybridization of grasses and cereals. The following subjects are be discussed: 1) methods of somatic embryo production, 2) levels and methods used for somatic hybrids' description 3) species participating in hybridization programs, 4) traits used for selection of somatic hybrids in in vitro culture, 5) agricultural traits searched in particular combination of hybridization, 6) ploidy of somatic regenerants and their isoenzymatic variation.
The results published in recent years proved that Agrobacterium based system for genetic transformation was also suitable for cereal crops. Several groups were able to obtain transgenic rice, corn, wheat and barley using hipervirulent strains Agl1 and EHA101 (or EHA105) or 'regular' LBA4404 strain with superbinary vector pTOK233. The first phase of our research was designed to establish transformation susceptibility of two wheat, two barley and one triticale cultivars using three different bacterial systems. Two of those systems were based on hipervirulent strains: Agl1 (pDM805) and EHA101 (pGAH). The third one combined strong virulence of pTOK233 vector and commonly-used LBA 4404 strain. Putative transgenic plants (regenerated and rooted under selective pressure of appropriate factor and further confirmed with GUS or PCR) were obtained for barley (cultivar Scarlett), wheat (Torka and Kontesa) and triticale (Wanad) with Agrobacterium strain Agl1. Kontesa's putative transgenics were also obtained with the strain EHA101. The highest rate of selection of putative transgenics was for Agl1 / phosphinothricine and ranged from 9 to15% for wheat cultivars. The lowest rate for the same strain and selection was 0,5% for barley cv. Scarlett.Inoculation of 700 immature embryos of barley cv. Lot with three bacterial systems (strains, vectors and selection factors) failed to produce putative transformants. Also no putative transgenics of barley Scralett, wheat Torka and triticale Wanad were obtained after transformation with EHA101 and selection on higromycine. Selection with kanamycin and hygromycin + kanamycin after transformation with EHA101 and LBA4404 respectively also failed to give positive results.
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