Assessment of Age-Related Polyploidy in Quercus Robur L. Somatic Embryos and Regenerated Plants Using DNA Flow Cytometry

Flow cytometric analysis with 4,6-diamidino-2-phenylindole (DAPI) staining was used to screen for chromosomal changes in Quercus robur during in vitro culture. The initiated cell lines (1992 until 1999) were maintained via secondary embryogenesis on P24 medium with 0.9 M 6-benzylaminopurine (BAP) in regular subculture intervals of 6 weeks. Regenerated plants established in the greenhouse and in vitro plantlets derived from encapsulated somatic embryos were screened. The embryogenic cell lines were characterized as individual clones by isoenzyme analysis. Flow cytometric relative DNA content analysis of the first screening period revealed that somaclonal variation in form of tetraploidy occurred in two out of 26 tested somatic embryo clones (Alt and Jung). These two clones lost their ability to convert into plantlets. Intraspecific relative DNA content variation including technical variation was below 3 %. In the second screening period, however, 3 out of 37 clones (Alt, E4.31H9 and P3.27H) contained tetraploid cells leading to the assumption that the frequency of tetraploidy seems to be correlated with the duration of in vitro culture. No chromosomal differences were detected in regenerated plants. However, tetraploidy occurred in 8 % of the tested clones over a culture period of 7 years.     

Detection of microsatellite instability during somatic embryogenesis of oak (Quercus robur L.)

Five microsatellite loci (QpZAG1/5, QpZAG9, QpZAG36, MSQ4, MSQ13) were used to test for genetic stability of three somatic embryogenic culture lines of Quercus robur L. and plantlets derived therefrom. DNA variation was detected among somatic embryos within all embryogenic lines, whereas no genetic instability was found among the regenerated plants. Two microsatellite loci revealed variation, and a locus-dependent instability was observed. The most polymorphic and useful microsatellite locus for detecting genetic variation was QpZAG9, with 28.5% of the investigated loci being variable.     

Development and germination of Abies alba somatic embryos

Mature zygotic embryos of Abies alba mull were placed on a modified MCM medium (basal medium-BM) with 2.2 M benzyladenine and 2.3 M kinetin to induce embryogenic suspensor masses (ESM). These ESM proliferated on induction medium supplemented with 0.2 M 2,4-dichlorophenoxyacetic acid. From 61 ESM lines induced, 36 are still in culture after 2 years, of which 18 show embryogenic potential indicated by spontaneous formation of globular somatic embryos on the proliferation medium supplemented with 500–1000 mg l^-1 casein hydrolysate and 500 mg l^-1 l-glutamine. ESMs from cell line 2/56 were conditioned 1 week on BM with 58 mM sucrose and 10 g l^-1 activated charcoal for maturation of somatic embryos. Maturation was achieved on BM containing 20 M (±)cis-trans-abscisic acid in combination with 111 mM maltose. Organic nitrogen supplements improved the proliferation rate of cell line 2/56 as well as the maturation and vitality of the somatic embryos. Partial drying was necessary for subsequent root development. Plantlets with a root, primary needles and a terminal bud developed on BM when a combination of 30 mM sucrose and 50 mM maltose was provided as carbon source.Abbreviations BM basal medium - BA benzyladenine - ESM embryogenic suspensor mass - 2,4-d 2,4-dichlorophenoxyacetic acid - CH casein hydrolysate - l-gln l-glutamine - ABA (±) cis-trans-abscisic acid     

Embryogenesis and genetic stability in long term megagametophyte-derived cultures of larch

Embryogenic cultures derived from megagametophytes of Larix decidua were maintained for up to 17 years. A few lines were divided into sub-lines, which were maintained in the same manner as the others. Embryogenic tissue was grown on 1/2 strength LM medium supplemented with glutamine and casein hydrolysate at constant temperature and light regimes. Chromosome counts were conducted at various times. DNA content was assessed by flow cytometry. Embryogenesis was monitored with each transfer and records of all appearances of green mature embryos were kept. Chromosome number was found to vary. DNA content and chromosome number, both of which had doubled a number of years after initiation, stabilized around 24 chromosomes for most cultures. A few lines showed substantial increases in chromosome number. One of these lines lost vigour and died. Another line showed a further doubling of DNA content. No lines were embryogenic over the entire period. Embryogenicity was lost completely in some lines, but in others the loss was temporary, as periodic restoration of embryogenesis was observed.