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1

Agrobacterium-mediated transformation of plants - basic model of the process and key factors involved in it

100%
Nadolska-Orczyk A.
Biotechnologia
|
1999
|
issue 1
116-124
EN
During infection of plant tissue by Agrobacterium, a fragment of Ti plasmid (T-DNA) is transferred to a cell. T-DNA is delimited by 25 bp direct repeats, which are necessary to direct it. Any piece of DNA flanked by these sequences can be transferred to the plant cell, where it becomes integrated into the plant genome. The processing and transfer of T-DNA are mediated by vir region localized on the Ti-plasmid. Based on the summarized model of plant transformation via Agrobacterium, the key factors involved in this mechanism are reviewed. There are two basic components of the process: Agro strain with its vector system and recipient, usually totipotent plant cell. The most important factors, which should be taken into consideration, include: Agrobacterium host specificity and its vector system, tissue culture techniques and their limitations, chemical agents inducing virulence genes and selection strategies. Some of them can influence the expression pattern of the introduced genes.
2

Transgene expression and gene silencing in cereals

100%
Nadolska-Orczyk A.
Biotechnologia
|
2006
|
issue 4
168-180
EN
Genetic transformation of cereal crops is a powerful research tool for analysis of gene function and varietal improvement. Application of the method is possible when the expression of introduced transgene is on the desired level and stable over several generations. The production of transgenic cereals was mainly performed by microprojectile bombardment. However, some advance was also achieved by application of Agrobacterium-mediated transformation. For rice, which is the cereal model species, this method is routinely used, while for many others, especially polyploids, it has been developed very recently and only in a few laboratories. We still lack the knowledge whether the main features of Agro-mediated transformation (i.e. integration of one or few copies usually not rearranged and well defined transgene cassette) influence the transgene expression in polyploid cereal species. This review discusses known mechanisms possibly involved in transgene silencing, using both transformation methods. Part of the discussion is focused on transgene expression / silencing in relation to large genomes of polyploid cereals. Another application of genetic transformation, based on RNAi technology (RNA interference), is silencing of selected genes. This could be used to study gene function as well as to induce silencing of the native, single or family genes of cereals. Two strategies of silencing are discussed: a strategy of transcriptional gene silencing (TGS) and posttranscriptional gene silencing (PTGS).
3

Mechanism of Agrobacterium-mediated genetic transformation of plant cell

100%
Nadolska-Orczyk A.
|
EN
Agrobacterium-mediated genetic transformation is the only known example of horizontal gene transfer from bacteria to eucaryota including plants, fungi and animal cells. The knowledge of the basic mechanism of this process is the key to understand problems concerning methods of plant transformation and transgene expression. The main element of the system is Ti plasmid (tumor-inducing) containing T-DNA (transferred DNA) delimited by 25. nucleotide sequences (left and right borders). Any DNA located on Ti plasmid and flanked by these two borders might be recognised by Agrobacterium as a T-DNA and integrated into plant chromosome. The process is controlled by ten vir operons located on Ti plasmid. The most important products of the vir genes are VirA and VirG controlling the expression of all the other vir genes. VirD1 and VirD2 recognise 25 bp border sequences and take part in endonucleolitic cleavage. Additionally, VirD2 covalently attached to the 5?-end of single stranded (ss) T-DNA targets it into the nucleus of a plant cell. T-strand is coated by VirE2 molecules, each containing two sites of nuclear localisation signals (NLS). Eleven of VirB and VirD4 proteins are required to form a transmembrane channel and transfer T-strand to the plant cell. Some genes of the bacterial chromosome are responsible for bacterial attachment and colonisation of the plant cell.
4

Plant biotechnology and environment ? possibilities, benefits and potential risk

63%
Nadolczyk-Orczyk A., Nadolska-Orczyk A.
Biotechnologia
|
2002
|
issue 4
227-238
EN
Plant biotechnology is the key to solve most of the problems of modern agriculture. Today, genetically modified plants are planted on half of the acres of USA and it is expected that in 2030 most of cultivars will be transgenic. On the other hand, this technology has raised public?s fears. This article presents the possibilities of applying biotechnology to environmentally safe agriculture. It also discusses the potential risk and benefits of releasing the GMO into the environment.
5

The determinants of grain texture in cereals

51%
Nadolska-Orczyk A., Gasparis S., Orczyk W.
Journal of Applied Genetics
|
2009
|
vol. 50
|
issue 3
185-197
EN
Kernel hardness is an important agronomic trait that influences end-product properties. In wheat cultivars, this trait is determined by the Puroindoline a (Pina) and Puroindoline b (Pinb) genes, located in the Hardness locus (Ha) on chromosome 5DS of the D genome. Wild type alleles code puroindoline a (PINA) and puroindoline b (PINB) proteins, which form a 15-kDa friabilin present on the surface of water-washed starch granules. Both the proteins are accumulated in the starch endosperm cells and aleurone of the mature kernels. Puroindoline-like genes coding puroindoline-like proteins in the starch endosperm occur in some of the genomes of Triticeae and Aveneae cereals. Orthologs are present in barley, rye and oats. However, some genomes of these diploid and polyploid cereals, like that of Triticum turgidum var. durum (AABB) lack the puroindoline genes, having a very hard kernel texture. The two wild type alleles in opposition (dominant loci) control the soft phenotype. Mutation either in Pina or Pinb or in both leads to a medium-hard or hard kernel texture. The most frequent types of Pin mutations are point mutations within the coding sequence resulting in the substitution of a single amino acid or a null allele. The latter is the result of a frame shift determined by base deletion or insertion or a one-point mutation to the stop codon. The lipid-binding properties of the puroindolines affect not only the dough quality but also the plants' resistance to pathogens. Genetic modification of cereals with Puroindoline genes and/or their promoters enable more detailed functional analyses and the production of plants with the desired characteristics.
6

Agrobacterium-mediated genetic transformation of cereal crops

51%
Nadolska-Orczyk A., Przetakiewicz A., Orczyk W.
Biotechnologia
|
2000
|
issue 4
93-98
EN
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.
7

Application of RNA interference for cereal crops biotechnology

51%
Nadolska-Orczyk A., Zalewski W., Gasparis S., Orczyk W.
Biotechnologia
|
2010
|
issue 3
53-63
EN
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.
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