Jokioinen, Finland
March 16, 2009
Finnish rye bread now usually
made with imported rye
Healthy dark rye bread is a staple of the Finnish diet, but
nowadays most of the rye used by bakeries is imported from other
countries. Rye is less widely cultivated in Finland than other
crops because the subsidies are low and the plant is sensitive
to weather conditions, meaning that the crop yield is often
small and baking quality poor.
Teija Tenhola-Roininen, Research Scientist at
Agrifood Research Finland (MTT)
constructed a linkage map of rye in her doctoral research and
found DNA markers that can be used to breed rye lines that are
better adapted to the Finnish climate.
The DNA markers she discovered will help select lines with a
short straw and a good pre-harvest sprouting resistance. A short
straw is beneficial because it protects the stalks from being
beaten down by late summer rainfall. Pre-harvest sprouting
resistance is important because sprouting before harvest reduces
the falling number of rye and decreases the quality of the grain
for the baking industry.
Doubled haploid has no secrets
Tenhola-Roininen used homozygous doubled haploid plants in her
research. The two homologous chromosomes in these plants are
identical. In natural conditions, rye is cross-pollinated and
has two different homologous chromosomes, one from each parent.
"The advantage of using doubled haploids in plant breeding is
that all their characteristics are manifest in the plant, making
selection easier. In normal cross-pollinated rye some of the
characteristics are recessive and therefore cannot be detected,"
Tenhola-Roininen explains.
Doubled haploids allow researchers to speed up the breeding
process of a new rye line by 3 - 4 years.
Research plants produced by anther culture technique
Tenhola-Roininen produced the doubled haploids using the anther
culture technique. This is a process whereby the anthers are
plucked from the florets and placed on a growth medium to allow
new plants to grow from the microspores contained in the
anthers.
Doubled haploid rye plants are produced by the anther culture
technique either spontaneously or haploid plants can be treated
with colchicine to duplicate their chromosomes. During the
research project, doubled haploid formation was also promoted by
applying cold stress to the plants and heat stress to the
anthers.
In some breeding lines these treatments increased the
spontaneous regeneration of doubled haploids while in others
they reduced it. In certain lines, the most effective treatment
proved to be storing the spikes of rye at 4 °C for a period of
three weeks.
"At least 10% of the green plants regenerated by anther culture
were suitable for breeding. Generally the mortality rate of the
plants generated by anther culture was high, a fact that will
have to be taken into consideration when planning the use of rye
doubled haploids in further research," Tenhola-Roininen points
out.
DNA markers found on chromosome 5R
In order to find DNA markers, two rye populations were generated
with various degrees of dwarfism (short straw) and pre-harvest
sprouting resistance. The indicator used to measure pre-harvest
sprouting was the alpha-amylase activity of grains, which
correlates negatively with the falling number.
To find markers which indicated pre-harvest sprouting
resistance, Tenhola-Roininen constructed a genetic map
containing 281 DNA markers. It is the first genetic map of rye
constructed using doubled haploid plants in the world. She
discovered that one genetic region associated with sprouting
resistance is located on chromosome 5R.
In addition to arbitrary DNA markers, Tenhola-Roininen studied
DNA markers located on 5R because a dwarfing gene is known to be
located on that chromosome. She developed a DNA marker which
breeders can use to select the individuals with the short straw
from their breeding material with an error margin of 13%. This
DNA marker is now being tested in rye breeding.
The doctoral thesis by Teija Tenhola-Roininen, M.Sc. "Rye
doubled haploids - production and use in mapping studies" will
be publicly examined at the University of Jyväskylä on 27 March
2009. Professor Teemu Teeri of the University of Helsinki will
serve as the opponent and Professor Jari Ylänne of the
University of Jyväskylä will serve as the custos.
Related December 2007 article from
Koelypsy magazine
Gene
transfer technology ready for barley
MTT Agrifood Research Finland has adapted an agrobacteria-based
genetic transfer technology for barley for its own use.
Development of gene transfer methods forms part of the
Disease-Resistant Barley project, which provides genomic tools
for managing and monitoring barley net blotch.
Net blotch is the number one disease affecting barley in
Finland. Genetic transfer will enable us to examine which genes
block net blotch in barley.
- Once we have identified the gene, we can create breeding tools
to screen that particular property from wild barley crops, for
example. This can then be introduced to new breeds of barley
through a more efficient backcross breeding programme, explains
Outi Manninen, the researcher in charge of the project at MTT.
Next year, MTT will witness the start of research funded by the
Academy of Finland, where the objective is to locate and isolate
the genetic factor that blocks net blotch infection in barley.
At a later stage, this resistance gene will be transferred to
barley varieties susceptible to the disease in order to monitor
its function.
For the purposes of the Disease-Resistant Barley project, the
genetic transfer has been implemented using a foreign sample
barley called "Golden Promise". The same agrobacteria
transmission method will not necessarily succeed in all types of
barley.
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MTT archives
According to Eeva-Liisa Ryhänen, Director of Biotechnology and
Food Research at MTT, genetic transfers of barley represent a
major step by MTT in genetic transfer research involving plants.
The use of genetic transfer technology is on the increase, and
she considers it important that the research centre monitors its
development and masters the new methods.
- In our research involving genetically transferable plants our
focus is particularly geared towards improved disease resistance
and health benefits, Ms Ryhänen says.
She emphasises that the development of gene transfer technology
is not only aimed at GM breeds but that it is also useful in
investigating genetic function.
Experience from England
The project, which started in 2005 and runs until 2008, is
implemented through a partnership between MTT, the University of
Helsinki and Boreal Plant Breeding Ltd. In addition to genetic
transfer tools, the project has also developed other genomic
applications for barley.
The use of genomic tools will require close international
cooperation. Veli-Matti Rokka, Senior Researcher at MTT, has
studied the genetic transfer of barley at the John Innes Centre
and the Scottish Crop Research Institute in Great Britain, and
has subsequently trained other researchers at MTT.
According to Mr Rokka, Finland is advanced in barley research.
- MTT's plant genomics research team is of a high international
standard. The team has also gained expertise in extensive
on-farm cell research projects, which have been carried out by
the research unit not only in respect of barley but also with
oats, wheat, rye, potatoes, oil plants and raspberries.
New application in use
Genetic transfers in the Disease-Resistant Barley project are
implemented through the retrotransposon promoter areas of
barley, which have been isolated by Professor Alan Schulman and
his team at MTT.
Genetic promoters consist of areas of DNA that control the
activation of genes in a specific area of a plant or at a
specific stage of its development. Retrotransposons, like genes,
are hereditary, and both have their own promoters.
Professor Schulman has been carrying out pioneering
retrotransposon research for almost 20 years.
- If too many genetic copies are transferred at a time, the
promoters usually "switch off". We believe that we can overcome
this problem by using retrotransposon promoters to assist in
genetic transfer, since they are naturally present in thousands
of copies.
The promoter areas used in the transfers may originate either
from the same plant, a different plant or from a virus, for
example.
- Our method is a brand new approach to the genetic modification
of plants. It is also directly applicable to the gene transfer
of any other cultivated plant. Moreover, it has other possible
uses within genomics research which, for example, seeks to
improve the function of genes, Professor Schulman explains.
Agrobacteria used in the transfer
The MTT plant genomics team employ agrobacteria as agents in
gene transfer. There are other means and, with barley, using
bioballistics is another significant genetic transfer method.
- For the moment, however, agrobacteria-based genetic transfer
is more effective, as the gene will usually transfer to the
plant in just one or two copies. If the volume of copies is too
high, genetic function may slow down, Veli-Matti Rokka explains.
Agrobacteria are a common form of soil bacteria, with a natural
capacity for gene transfer. In dicotyledonous plants they cause
galls to form in any damaged areas. This disease will not
develop in barley under natural conditions, and the properties
of the disease have been completely removed from the bacteria
base used in laboratory settings.
- Each bacterium contains a ring-like DNA molecule, to which the
transfer gene will be attached, and which will then carry it to
the recipient cell. The gene that we transfer in this project is
a marker gene that produces fluorescent protein. Furthermore,
the molecule carries a hygromycin resistance gene, which is used
in the laboratory to select the best cells for genetic transfer,
Mr Rokka explains.
Transfer to the seed embryo
Genetic transfer is carried out with immature barley seed
embryos. For this reason, the embryos must be detached whole
from raw seeds and at precisely the right stage; this is the
most laborious phase of the gene transfer process.
The next stage is introducing the bacteria to the embryo. The
embryonic cells will be grown on a base containing antibiotics,
and only cells with natural resistance to hygromycin will
survive.
- MTT research has shown that all plants produced from embryonic
cells have proved genetically transferable. The method has been
developed to a sufficient standard and it works well, explains
Outi Manninen.
However, the entire process from normal barley to a finished GM
plant takes almost a year.
According to Ms Manninen, selection by antibiotics has been
broadly used in genetic transfer research as well as in the
production of old GM plant breeds. Thanks to the new technology,
the property of resistance to antibiotics can be removed from a
new plant at a later stage, and this is already a compulsory
requirement for new production plant breeds.
Tight controls
GM barley is grown in MTT laboratories and greenhouses
separately from other plants. Outi Manninen says that the
Disease-Resistant Barley project has also created an opportunity
to train the members of MTT's plant genomics team in the special
requirements of handling GM materials.
- The regulations governing GM plants are extremely strict. We
must strive to keep a number of different risks at a minimum at
every stage of the process. Not a single GM seed can be allowed
to escape into the natural environment.
text: Päivi Haavisto
photo: Yrjö Tuunanen |
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