Somatic cell genetics in larches (Larix spp.)

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dc.contributor.author Pattanavibool nee Vongvijitra, Rungnapar
dc.date.accessioned 2017-05-18T21:17:04Z
dc.date.available 2017-05-18T21:17:04Z
dc.date.copyright 1996 en_US
dc.date.issued 2017-05-18
dc.identifier.uri http://hdl.handle.net/1828/8141
dc.description.abstract Studies of somatic cell genetics in larches (Larix spp.) were carried out using somatic hybridization, cytogenetics as well as fluorescence in situ hybridization. Haploid embryogenic protoplasts are ideal sources for somatic hybridization if they possess stable chromosome complements. In my protoplast fusion experiments, I used diploid embryogenic protoplasts because genetic variation was detected in the haploid lines available. Cytogenetics coupled with fluorescence in situ hybridization was used to reveal genetic instabilities in haploid embryogenic lines as well as to produce a standard karyotype of Larix decidua. A diploid embryogenic culture of tamarack (Larix laricina, line L2) was used as one of the fusion partners while the other partner used was one of the two hybrid larches (Larix x leptoeuropaea, line L5 and Larix x eurolepis, line L6 ). The selection system was based on complementation of metabolic inhibition (with sodium iodoacetate) of tamarack and the lack of ability to produce mature embryos of the hybrid larches. Ideally, only the heterofused cells would have been able to regenerate. The vital fluorescent dyes, DiOC₆ and R6 , were used to stain protoplasts of each parent to determine fusion events and frequencies. I compared fusion firequency as well as cell division between fusion mediated by PEG or electric pulses. PEG-mediated fusion resulted in 14-18 % of heterofused cells. All electrofusion treatments gave much lower fusion frequencies, at only 4-8 %. Although the percentages of cell division after 4d of PEG-fusion (17-24%) and electrofusion (19.3%) were about the same, PEG-fusion was found to be a more efficient means than electrofusion. Sodium iodoacetate at a concentration of 4-5 mM was found to efficiently inactivate the protoplasts of tamarack. All control-treated protoplasts as well as mixed cultures (unfused protoplasts) died. Tamarack protoplasts produced mature single embryos, whereas protoplasts of hybrid larches never completed embryogenesis. Some post-fusion products produced colonies and mature embryos. RAPD was used to verify the hybridity of those fusion-derived colonies and mature embryos. Of thirty-one fusion experiments between lines L2 and L5, only one produced individual colonies. Of the thirteen colonies which developed in that experiment, none yielded mature embryos. RAPD analysis of the colonies picked out from L2/L5 fusion showed DNA banding characteristics of L5. From twenty four experiments fusing L2 and L6 , there were five experiments which produced colonies. A total of two hundred and thirty nine individual colonies and nineteen single mature embryos were picked out from those L2/L6 fusions. RAPD banding profiles of eighty seven colonies and nineteen mature embryos showed DNA banding characteristics of L2 only. Tested haploid embryogenic lines (total of 6 lines; n=12) of Larix decidua initiated from megagametophyte tissue were maintained on half-strength Litvay’s medium without growth regulators. All lines had been verified as being haploid by chromosome squashes when they were initiated. Some lines have been stably haploid for only a short period of time while others have been stable for many years. Variations in chromosome numbers increased proportionately with the age of the culture. Haploids doubled their chromosome numbers. Aneuploidization occurred because of unequal separation of the chromosomes. Unusual events during mitosis such as formation of anaphase bridges, fragmentation of chromosomes, and development of long kinetochores were detected. There was a tendency of rising chromosome numbers in all lines tested over the years. Fluorescence in situ hybridization (FISH) was used to physically map highly repetitive sequences of genes coding for 18S-5.8S-26S rDNA on Larix decidua chromosomes. A karyotype of L. decidua (2n=24) was created from average relative lengths derived from the six best squashes with strong probe-target FISH signals. Hybridization of 18S-26S rDNA onto L. decidua chromosomes gave very precise locations of secondary constriction as well as unexpressed nucleolar organizer regions. In L. decidua, there were 6 major 18S-26S rDNA loci detected in 60.53% of cells (23 out of 39 cells). Five I8S-26S rDNA loci were also found but at a lower rate of 39.47%. All loci were expressed and located at the sites of secondary constriction on chromosomes 2, 4 and 7. Two extra locations of 18S-26S rDNA were mapped on aneuploid chromosomes (30 chromosomes) derived from cells of an aneuploid line (line 2110) of L. decidua. Chromosome measurement resulted in a preliminary karyotype of this line. The relative total lengths and locations of I8S-26S rDNA of standard (2n=24) chromosomes and aneuploid (2n=30) chromosomes was compared. en_US
dc.language English eng
dc.language.iso en en_US
dc.rights Available to the World Wide Web en_US
dc.subject Somatic cells en_US
dc.subject Chromosomes en_US
dc.subject Cytogenetics en_US
dc.subject Plant genetics en_US
dc.title Somatic cell genetics in larches (Larix spp.) en_US
dc.type Thesis en_US
dc.contributor.supervisor Aderkas, P. von
dc.degree.department Department of Biology en_US
dc.degree.level Doctor of Philosophy Ph.D. en_US
dc.description.scholarlevel Graduate en_US

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