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Plant Physiology
B. Panis
and S. Carpentier
Plant physiologists investigate physiological processes at different
levels of complexity, from molecules to complete organisms, involving
methods and techniques from biochemistry, molecular biology, biophysics,
cytology, anatomy and genetics. Currently, emphasis is most often put
on the molecular and ecological aspects in plant science. The link between
these two disciplines, i.e. plant physiology, is often neglected. The
Laboratory’s extended expertise on in vitro techniques
(somatic embryogenesis, protoplast culture, meristem culture and cryopreservation),
together with chromatographic analysis and techniques like 2D PAGE analysis
for proteomic research is used to unravel processes in plants.
Somatic embryogenesis
(B. Panis)
 Embryogenic
cell suspensions (ECS) are the material of choice for rapid multiplication
and plant improvement programs relying on in vitro mutagenesis
and genetic transformation.
Especially the single cell origin of the regenerated plants makes
them valuable material for regeneration studies and genetic engineering.
Although techniques for somatic embryogenesis in banana are available,
the initiation of ECS cannot yet be considered a routine procedure
because of the low embryogenic response and the slow process. Only
0 to 20% of the inoculated explants respond and form embryogenic tissues.
The initiation of a banana suspension takes 9 to 26 months depending
on the type of explant and genotype. Needless to say that an optimization
of the existing protocols is a priority.
Useful links:
- Banana
and plantain embryogenic cell suspensions. INIBAP Technical Guidelines
- Banana
cell and tissue culture - review
About 20,000 plant species are believed to be endangered, rare and
threatened with extinction. Their preservation is essential for classical
and modern plant breeding programmes. Also, this biodiversity is a
source of compounds for the pharmaceutical, food and crop protection
industries. Conventionally, germplasm is conserved through seeds,
roots, cuttings, etc. Cryopreservation or freeze-preservation at ultra-low
temperature (-196°C) is the method of choice for the long-term
conservation of plant germplasm, since biochemical and most physical
processes are arrested under these conditions. As such, plant material
can be stored for unlimited periods.
A manual describing the different cryopreservation protocols for banana
tissues is available online.
In December 2006, Dr. B. Panis became chair of the EU
COST Action 871: “CRYOPLANET, Cryopreservation of crop species
in Europe”. In this Action, researchers belonging to 16
European countries will collaborate and hold meetings on different
aspects of Plant Cryopreservation. Two working groups are distinguishes
within the project, i.e. (i) fundamental aspects of cryopreservation/cryoprotection
and genetic stability and (ii) technology, application and validation
of plant cryopreservation. The Action will run until December 2010.
Cryopreservation of meristem cultures (including routine cryopreservation)
(B. Panis)
In vitro proliferating shoot-tips are currently maintained
under slow- growth conditions at reduced temperatures and light intensity
(International
Transit Centre). Although this mode of preservation has proven
its value, somaclonal variation, loss of morphogenic potential, contamination,
etc. remain serious impediments to the conservation of in vitro
collections. Moreover, the maintenance of such a large collection
is labour intensive. Therefore, to complement the active collection,
a base collection using cryopreservation is under development. During
the last decades, 3 efficient cryopreservation protocols for meristem
cultures of banana were established at the Laboratory. These
are now routinely applied to the banana collection (supported
by INIBAP, Gatsby Charitable Foundation and the World bank).
Currently (situation on 1 February, 2010), 760 Musa accessions
are now considered successfully cryopreserved with each at least 3
independent successful repetitions. This cryopreserved collection
covers a wide variety of banana land races belonging to all genomic
groups, hybrids as well as wild diploid species..
Cryopreservation of cell suspensions
(B. Panis )
Until now, embryogenic cell suspensions ( somatic
embryogenesis) are essential for genetic engineering of Musa spp.
(see molecular biology group). The initiation of transformation competent
banana embryogenic suspensions is still difficult and time-consuming.
Moreover, once initiated, they are subject to somaclonal variation,
loss of regeneration capacity and contamination. Their safe, long-term
storage through cryopreservation is therefore essential.
Currently, more then 1,000 cryotubes containing transformation competent
cells from different banana cultivars are safely stored in liquid nitrogen.
Fundamental aspects of cryopreservation
(B. Panis, S.
Carpentier and J. Geuns)
 Cryopreservation
procedures are mainly developed on empirical grounds. Hence, cryopreservation
of plant germplasm is still not standardised and depends on the tissue
and plant species/variety. We aim to determine the necessary physico-biochemical
state of any plant species/tissues needed for successful cryopreservation.
As such, a generally applicable cryopreservation protocol can be established
and becomes available for all germplasm collections. For this, methods
for Differential Scanning Calorimetry (DSC) analysis and analysis
of sugars, polyamines, transglutaminases, proteins, sterols, membrane
lipids, cytoskeletal changes and oxidative stress are investigated
to plant tissues that are subjected to cryoprotection. Results are
correlated with post-thaw viability rates. As such, new/adapted cryopreservation
protocols can be developed. This study is carried out with financial
support from the Commission of the European Communities, in collaboration
with partners from Italy, Germany, France and UK. More information
on the CRYMCEPT project is available on
line.
Several abiotic environmental situations (e.g. drought, low temperature,
high salt content)
provoke plant dehydration, causing growth retardation or even death.
Water deficit is one of the major causes of yield loss worldwide.
It starts as soon as the water demand exceeds the water supply. The
knowledge of the underlying molecular reactions in the osmotic stress
models will be implemented for crop improvement.
“Proteomics is the endeavour to understand gene function and
to characterize the molecular processes of the living cell through
the large-scale study of proteins found in specific biological contexts”
(Liska and Shevchenko, 2003). Two dimensional gel electrophoresis
is quantitatively a strong and reliable technique and it is the most
suitable profiling approach for species of which the genome is not
(fully) sequenced. Analyzing directly the end product (proteins in
stead of RNA) approaches more the true physiology and ensures thanks
to the well characterized (model) organisms and tandem mass spectrometry
an easy and reliable identification of the significant proteins. The
identification of the proteins is realized in collaboration with the
Laboratory of Plant Biochemistry and Physiology at the University
of Antwerp and with the Laboratory for Protein Biochemistry and Protein
Engineering at the University of Gent. In order to facilitate future
proteome analysis on meristematic cells and to make the data accessible,
an on-line
database was constructed.
Useful links:
Optimized
Proteomic Methods to Unravel Biochemical Processes in Banana Meristems
during In Vitro Osmotic Stress Acclimation (Ph.D. Sebastien Carpentier,
2007)
Flanders
Proteomics
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