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					                  “Molecular and cytogenetic diagnostics in plant breeding”
                 School of Biotechnology, 27-30 April 2004, Poznań, Poland




       Strategies of exploring molecular markers linked to major genes
                     and QTLs important in plant breeding
                                           P. Masojć

  Agricultural University in Szczecin, Department of Genetics and Plant Breeding Sciences


The efficiency of the direct selection based on the phenotype evaluation is often very low due
to the strong environmental effects, multi-gene inheritance and inconvenient method of trait
detection. In these cases marker assisted selection (MAS), making use of molecular markers
linked with a gene of interest, can be applied. The most straightforward method for detection
of the selectable markers is bulked segregant analysis (BSA), performed mostly on F2
progeny of the cross between the two contrasting lines. However majority of traits of
agronomic importance are controlled by more than one gene and a more laborious method of
interval mapping (IM) is the best way to detect all markers necessary for selection purposes.
Application of interval mapping in rye led to detection of many QTLs for valuable traits and
linked to them markers. It can be shown that even with alpha-amylase activity, which is
highly sensitive to environmental changes, it is possible to dissect a trait into individual loci
and perform efficient MAS to detect genotypes accumulating favorable alleles.
      Although at the level of research any marker system that shows linkage with the trait of
interest is suitable, for practical breeding only not laborious, cheap and stably expressed
markers can be used. In fact, MAS planned on a large scale may be successful when STS,
SCAR or PCR-RFLP markers, separated in agarose gels, are available. Marker systems based
on electrophoresis in polyacrylamide gels are definitely not recommended for MAS. Thus,
marker conversion is usually performed at the final stage of marker development.
     It is often found that polymorphism in a marker locus can be explored for MAS only
within a single cross especially when outcrossing species is studied. In case of autogamous
crops rare marker allele can sometimes be associated with the positive trait across the wider
spectra of genetic materials. Generally it is advisable to make use of a very thight linkage
between a marker and a gene of interest. A good strategy is also identification of markers in a
good source material, having desired gene(s), which is already included into breeding
program. By doing this one can avoid long term effort to transfer valuable genes from low
yielding genetic materials to cultivars through repeated backcrosses and selection cycles.
Finding of marker tightly linked to a valuable gene may be a first step in a strategy of gene
isolation. Fine mapping combined with a database search for a candidate genes or screening
of appropriate DNA library can eventually open up a possibility of map-based cloning.




                                                                                               5
                  “Molecular and cytogenetic diagnostics in plant breeding”
                 School of Biotechnology, 27-30 April 2004, Poznań, Poland




                      Transgenic cucumber – what we learnt?
                                         S. Malepszy

Warsaw Agricultural University, Department of Plant Genetics Breeding and Biotechnology


Cucumber ( Cucumis sativus L. ) is an important crop, cultivated world-wide, mostly for fresh
consumption. It belongs to the most popular vegetables in Poland. Therefore the progress in
their genetics and breeding methods are very desirable. Obtaining the transgenic plants is one
of the modern methods for the studies of gene function and the improvement of economically
or environmentally important traits. In my group we are using this method for both
applications. In the lecture the most important results and experiences will be described
emphasizing the practical aspects.
      For the transformation of cucumber we developed the original regeneration method
from leaf microexplants. Using this method we obtained the transgenic plants with 9 different
constructs and we obtained the homozygous, transgenic lines. Presently we are working on
the new transformation procedure based on the cytokinin dependent suspension culture
characterized by the natural green fluorescence. This new method is marker free, highly
efficient and easy in identification of the transformation events in the single cells already.
      One of the constructs we introduced into cucumber to obtain a new practically important
trait contained the thaumatin gene deriving from West African shrub Thaumatococcus
daniellii Benth. The thaumatin is a protein constituted of 207 aa, responsible for sweet taste,
almost 10 000 times sweeter than sucrose on the molar basis and belongs to the GRAS
(Generally Recognized As Safe) group of compounds. We produced the homozygous lines
from 15 independent transformants and use them to various analyses, including: the 3 year
field trials to estimate the substantial equivalence; spatial and temporal protein accumulation
in plants; evaluation for consumption of fresh and processed cucumbers; feeding studies on
laboratory animals (experiments according to the “Novel Food” EU Directive); infection tests
with fungal pathogen Pseudoperonospora cubensis; metabolic profiles and chromosomal
localization of transgene using in situ hybridization.
      Another construct with a potential practical value that we introduced into cucumberis
the iaaM gene, coding for the IAA monooxygenase under the ovary specific promoter
pDefH9. The plants bearing the parthenocarpic fruits were obtained and homozygous lines are
currently bred. We studied also the activity and the inducibility of PR-2d tobacco promoter in
several transgenic cucumber lines. The high level of stable expression in the immature fruits
has been observed showing however the large interline differences.
      I would try to answer the following more general questions: 1. What really means a
“reliable and highly efficient transformation procedure” in cucumber; 2. Sweet protein in
cucumber – what we expect and what we have to obtain?; 3. Application of transgenics in
agriculture – what are the true limitations?




6
                  “Molecular and cytogenetic diagnostics in plant breeding”
                 School of Biotechnology, 27-30 April 2004, Poznań, Poland




              The potato with modified synthesis of 14-3-3 proteins
                                           J. SZOPA

           University of Wrocław, Institute of Biochemistry and Molecular Biology


The 14-3-3s constitute a family of highly homologous proteins, first discovered in brain tissue
and now thought to be present in all eukaryotic cells. Recently ten cDNAs in Arabidopsis,
seven in human cells and six in potato plant were found, all encoding highly homologous 14-
3-3 protein isoforms.
      Numerous recent investigations suggest the participation of the 14-3-3 proteins in cell
cycle control and gene expression. Members of the 14-3-3 protein family activate
neurotransmitter synthesis and ADP-ribosylation of proteins; regulate the activity of protein
kinase C, sucrose phosphate synthase, starch synthase and nitrate reductase. Moreover, they
display a phospholipase A2 activity and associate with the product of proto-oncogenes,
oncogenes and the cdc 25 gene. The broad spectrum of activities that are affected by 14-3-3
proteins suggests their potential in modifying the plant development and metabolism.
      While there is substantial progress in the identification of diverse partners of 14-3-3 in
recent years, at least two important questions need to be answered. Is there any specificity
within 14-3-3 isoforms in the binding of diverse partners? Does this binding affects plant
metabolism or physiology in vivo?
      The significance of 14-3-3 protein in potato metabolism has been shown by the use of
transgenic plants in which 14-3-3 protein has been either increased by the expression of a
Cucurbita pepo cDNA or decreased by an antisense RNA method.
      It was found that overexpression of 14-3-3 protein induced an increase in the content of
catecholamine and soluble sugars in leaves, and a reduction of tuber size and starch content in
greenhouse plant.
      The repression of 14-3-3 synthesis resulted in opposite effect. All the repressed plants
showed significant increases in nitrate reductase (NR) activity, suggesting that the regulation
of NR occurs in vivo, and is not isoform dependent. The increase in NR activity resulted in a
significant increase of selected amino acids (methionine) content and also total protein level.
Thus, the NR activity is negatively correlated to the 14-3-3 level.
      The level of sucrose phosphate synthase (SPS) activity is also significantly increased in
all the 14-3-3 underexpressed transgenes, and remarkably, the increase in enzyme activity is
accompanied by respective changes in carbohydrate level in the tubers. Likelihood NR, the
activity of SPS is negatively correlated to 14-3-3 protein level. Also the starch content is
negatively correlated to 14-3-3 protein level and catecholamine quantity in transgenic tubers.
It was thus proposed that 14-3-3 protein affect the carbohydrate metabolism in potato via
regulation of respective enzyme activities and catecholamine synthesis.
      To answer the question on isoform specificity, the isoforms gene promoters were first
analysed for specific domains content by the comparing to the known sequences accumulated
in database. Then the promoter characteristic was studied in transgenic plants transformed
with reporter GUS gene under the control of the 14-3-3 promoter. The data obtained strongly
suggest that the function of particular isoform at least partially derives from promoter
specificity.



                                                                                              7
                  “Molecular and cytogenetic diagnostics in plant breeding”
                 School of Biotechnology, 27-30 April 2004, Poznań, Poland




               Molecular cytogenetics – possibilities and prospects
                       of FISH-ing plant chromosomes
                                         R. HASTEROK

              University of Silesia, Department of Plant Anatomy and Cytology


Modern cytogenetics offers a wide spectrum of techniques that are useful in plant genome
analysis and that can potentially be exploited in plant biotechnology and breeding programs.
At present, one of the most widely used is a molecular cytogenetic method known as
fluorescence in situ hybridisation (FISH) which, in short, is based on the mechanism of
kinetically controlled renaturation of labelled DNA molecules called probe DNA with
complementary DNA substrates in cytological preparations. In recent years, rapid progress
has been made in the development of different technical aspects of FISH. There is a growing
number of sequences that can be physically mapped in situ on chromosomes of different plant
species. New and more sensitive labels and detection systems, along with optimisation of
labelling and hybridisation procedures, allow generation of more efficient probes which can
be used, for example, in multi-target and multi-colour FISH experiments. Different kinds of
DNA substrates, ranging from metaphase chromosome preparations to extended chromatin
fibres, offer the choice of resolution of FISH signals that is most appropriate for a given
experiment. Last but not least, the introduction of more and more efficient and powerful
systems of fluorescence microscopy and image capturing extends the capabilities of
acquisition and analysis of results.
       In this presentation, the main areas of FISH exploitation will be presented, starting from
simple and mainly chromosome marker-oriented experiments based on rDNA probes towards
technically and/or scientifically more sophisticated ones like such as the use of ribosomal and
genomic DNA probes to determine the genomic constitution of ornamental lily hybrids and
addition lines of Brassica, and also in the molecular cytotaxonomy of some grass species.
Various sets of DNA probes, such as ribosomal, centromeric and telomeric, can also be used
effectively for other purposes, for example in cytological analysis of meiosis and mitosis.
Finally, some comparative genomic experiments will be presented, based on FISH of clones
obtained from bacterial artificial chromosome (BAC) libraries landed on chromosomes of
both monocot and dicot species.




8
                  “Molecular and cytogenetic diagnostics in plant breeding”
                 School of Biotechnology, 27-30 April 2004, Poznań, Poland




                    Harmonious Polish biotechnology foresight
                                       T. TWARDOWSKI

           Institute of Bioorganic Chemistry Polish Academy of Sciences, Poznań


The Polish market of 38 mln people is important for the United Europe. The economy and
scientific potential play important roles in the United Europe. We have to take these factors
into account. One can recognise three basic branches of modern biotechnology:
 Green, this is agrobiotech including the food industry;
 White, industry and biotechnics applied for the industrial production;
 Red, covering medicine with pharmacy, diagnostics and veterinary.

      The future prospects of biotechnology in Poland are connected with the conditions for
future development of national economy. Various factors influence the development of the
commercial biotechnology.
These are:
        – entrepreneurial attitude,
        – technical feasibility and infrastructure,
        – availability of capital,
        – legislation and regulations,
        – governmental initiatives in biotechnology,
        – public acceptance.

      The products of white biotechnology perfectly fit the needs of national economy and
will be welcomed by the public.




                                                                                              9
                   “Molecular and cytogenetic diagnostics in plant breeding”
                  School of Biotechnology, 27-30 April 2004, Poznań, Poland



                                Living medicine factories
                                  R. SŁOMSKI1,2, M. SZALATA 2
      1
          Poznań Agricultural University, Department of Biochemistry and Biotechnology
                  2
                    Institute of Human Genetics, Polish Academy of Sciences


The beginnings of genetic engineering date back to the 1970’s. Today this technology permits
us to produce animals that manufacture human proteins – one of the most expensive
medicines. Human proteins can even be made in bacteria, and this process is at present the
simplest, cheapest and quickest means of doing so. However, not all proteins can be obtained
in this way. This is why higher organisms – fungi, plants and animals – are also used in such
processes. In these systems, proteins are subjected to all the modifications needed for them to
function properly. Complete proteins can be obtained quickest though the use of mammalian
cell cultures. These, however, are extremely expensive, a factor that restricts their application.
Transgenic animals are produced by introduction of foreign genes into their cells. In order for
a gene to be transmitted to subsequent generations, it must be introduced into the reproductive
cells. The drawback of this solution may be the long time it takes for the transgenic organism
to develop. This is especially important in the case of livestock, e.g. pigs, sheep or cattle. If
the process of genetic modification was fully successful, however, then we can expect to see
the foreign proteins in the milk, urine, blood or semen of animals, and the eggs of poultry.
Recombinant proteins, also called fusion proteins, are obtained in genetic engineering by
attaching a sequence that encodes a “fused ” part to the DNA sequence that encodes a specific
protein. The additional piece increases the stability of the protein, facilitates and frequently
speeds up the isolation process. It is later removed via enzymatic processing. One of the
proteins whose production has gained a lot of attention recently is the growth hormone
necessary for the proper development of vertebrates. This hormone is involved in the
metabolism of mammals, stimulating the synthesis of proteins and the degradation of fats.
Attempts to introduce constructs containing the growth hormone gene into the genome of
animals were already made back in 1982. In the case of livestock, this approach was used to
try to accelerate their growth and to reduce the cost of breeding. Today such animals are
gradually being released onto the market.
       Until recently genetically modified cattle, sheep and pigs could only be made by directly
injecting new DNA sequences into a fertilized egg. Such pro-nuclear injection is inefficient
and gives no control over where in the chromosome the introduced gene is integrated. Often
multiple copies are introduced and both these factors cause highly unpredictable levels of
expression. Importantly pro-nuclear injection can only add genes. The ability to clone animals
from cultured cells by nuclear transfer means that it is now possible to introduce precise
genetic changes in livestock and genes have now been both introduced and removed to
animals using this technology. The development of efficient and reliable methods to allow,
for example, the deletion of both copies of a gene will require further improvements in
methodology and the extension of cell-based methods to other species, in particular birds,
involves major technical challenges.




10
                  “Molecular and cytogenetic diagnostics in plant breeding”
                 School of Biotechnology, 27-30 April 2004, Poznań, Poland




                Molecular markers – a new tool for an old science
                                       W. K. ŚWIĘCICKI

               Institute of Plant Genetics Polish Academy of Sciences, Poznań


Knowledge on a gene expression, a position effect, gene interaction as well as their chromosomal
localization is crucial for monitoring and improving a given organism. A knowledge on
linkages/distances between genes with agronomic importance and so called gene markers results in
practical advantages.
      In 1906, Bateson and Purnett described genetic linkages in Lathyrus odoratus L. Nearly
a century later, genetic linkage has become a very powerful tool for crop improvement. This
is largely due to the ability to develop molecular markers that detect DNA sequence
polymorphism and characterize their association and linkage with valuable alleles.
      The pea entered the genomic era with a well developed chromosome map having
numerous, easy recognisable markers. It resulted in the so called common map joining
classical as well as molecular and as an effect giving possibilities of using marker assisted
selection for many different characters of a cultivar ideotype. Molecular markers showed
clearly new chances for the QTL technique.
        Genes with a clear phenotype have played a key role in breeding different pea
ideotypes, e.g. a and r, le, af, i, u, p, v. Current knowledge of traits and molecular markers
suggests to use marker assisted selection in improving of resistance to diseases (e.g. powdery
mildew, fusarium wilt, viruses) as well as flowering and maturing time.




                                                                                             11
                  “Molecular and cytogenetic diagnostics in plant breeding”
                 School of Biotechnology, 27-30 April 2004, Poznań, Poland




        Secondary metabolites production using in vitro plant cultures
                                          W. GRAJEK

                     Poznań August Cieszkowski Agricultural University

Modern plant biotechnology includes such important applications as micropropagation,
construction and cultivation of transgenic crops, production of secondary metabolites using
cultures in vitro, and the use of cell cultures for chemical transformation. An important part of
these processes is performed using bioreactor techniques. The main field of cell and organ
cultures is secondary metabolite production and biotransformation. Numerous cell lines are
able to produce valuable chemical compounds which can be used in pharmaceutical industry,
as food additives, and as the preparations for cosmetic production.
Bioreactor cultures comprise different type of tissues including differentiated and
undifferentiated forms. The best-adopted form for bioreactor cultivation is suspension culture.
Usually cell density ranges from 103 cells/mL to 107 cells/mL, whereas the density of cell
aggregates amounts 103-105/mL. During last decades some specific methods allowing on log-
term organ culturing were also elaborated and scaling-up. Among the most important are
hairy root cultures which are especially efficient in secondary metabolite production.
        The key factors determining the high yield of secondary metabolite production are size
of inoculum, type and concentration of phytohormones added, source of nitrogen, sugar
concentration, medium aeration rate, viscosity of culture as an important factors in tissue
protection against mechanical stress, light intensity and osmotic pressure. The majority of
metabolites are produced in cells and accumulated in vacuoles what is a disadvantage from
technological point of view.
        Various treatments are applied in view to improve the yields of secondary metabolite
biosynthesis. An increase of metabolite production can be achieved due to elicitors addition
into culture medium, optimization of culture condition including physical and chemical
environmental parameters, supplementation of medium by some limited nutrients, and
exchange of growth medium during different growth phase. It should be stressed that various
interaction between different growth parameters are observed. An important factor of
metabolite production is choice of suitable bioreactor type. In some cases a special
construction should be used It is true especially for hairy root cultures.
        In plant cell cultures a serious problem is make separation and purification of
secondary metabolites due to their intracellular accumulation. To improve release of
metabolites different methods of treatments are proposed in order to increase the cell wall
permeability and extraction of chemical compounds. Among the most efficient the changes of
membrane potential using different polyelectrolytes, hydrolysis of phospholipids and
membrane proteins using enzymes, treatment with DMSO and SDS, thermal shocks, the use
of high hydrostatic pressure, extraction with organic solvents, changes in ionic strength and
osmotic pressure. These effects are illustrated with many examples of plant cultures in vitro.
A benefit effect has also immobilization of cells in hydrocolloids.
        Even many technological problems related to metabolite production using in vitro
cultures, some industrial applications are developed and successful development of this
technology is expected. For example, the bioreactor cell cultures are applied for production of
red pigment shikonin and some cytostatics, as taxol, vincristine and ajmalicine. Many other
secondary metabolites are the subjects of intensive investigations. Among them plant
pigments, enzymes, phytoalexins, antioxidants and aroma compounds can be mentioned.

12
 “Molecular and cytogenetic diagnostics in plant breeding”
School of Biotechnology, 27-30 April 2004, Poznań, Poland




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