Abstract
Differentiation in plant cell cultures was studied by
tissue culture and
genetic approaches. The influence of various
environmental factors on
differentiation was elucidated using Digitalis lanata
protoplasts in the first
part of the study. The molecular genetics of
differentiation were studied in
embryogenic birch (Betula pendula Roth.) cell cultures in
the second part.
Isolation and culture methods were developed for
Digitalis lanata seedling
protoplasts. The physiological state and the age of the
explant proved to be
critical for protoplast yield and viability. Suitable
osmotic pressure, high
protoplast density in the beginning of culture, gentle
enzyme treatment and
efficient purification procedures were essential for
protoplast regeneration.
In addition, the use of agarose as a gelling agent of the
medium was an
essential factor for the initiation and maintenance of
cell division. In
optimized conditions the first cell division occurred
after three weeks and
callus colonies were formed in two months. In order to
achieve embryogenesis or
organogenesis, the hormone contents of the culture medium
were changed.
However, abundant root formation was observed in all
cultures regardless of the
hormones used, whereas structures resembling embryos or
small shoots were
observed in only a few cases. This result suggests that
differentiation occurs
much earlier than previously believed, already in the
callus induction medium.
Thus, early markers are needed in order to identify
competent cells for
differentiation.
The BP8 gene was isolated from a birch genomic library by
heterologous
hybridization using a carrot embryospecific cDNA as a
probe. The BP8 gene was
sequenced and compared with the homologous carrot gene,
DC8. On the basis of
DNA sequence analysis the BP8 gene codes for a protein of
molecular weight 53
kDa and it exhibits 52% identity with the DC8 sequence at
the amino acid level.
On the basis of its structure and homology the BP8
protein belongs to the group
of LEA (late embryogenesis abundant) proteins. The
promoter regions of the BP8
and DC8 genes contained several homologous sequences.
Furthermore, similar
regulatory elements were found in both genes. The
expression of the BP8 gene
was studied in cell cultures of birch representing
different developmental
stages of somatic embryogenesis. The BP8 gene was shown
to be expressed at a
low level in proembryogenic cell aggregates. The low
expression level of the
BP8 gene does not encourage the use of this gene as a
molecular marker for
embryogenesis in birch cell cultures. The homology
between birch and carrot
genes suggests, however, that the methods described here
can also be used more
generally in order to identify early genetic markers for
differentiation in
other recalcitrant species.
A genomic library and six cDNA libraries of different
developmental stages of
birch somatic embryogenesis were constructed and stored
for future
investigation of differentiation and development in plant
cells.
Original language | English |
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Qualification | Doctor Degree |
Awarding Institution |
|
Supervisors/Advisors |
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Award date | 9 Dec 1994 |
Place of Publication | Espoo |
Publisher | |
Print ISBNs | 951-38-4643-1 |
Publication status | Published - 1994 |
MoE publication type | G4 Doctoral dissertation (monograph) |
Keywords
- plants (botany)
- tissue culture
- cell culture
- embryogenesis
- genes
- regulation
- markers
- protoplasts
- isolation
- ribonucleic acids
- deoxyribonucleic acids
- RNA
- DNA
- hybridization
- regeneration