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. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/8141 |
| Date | 18 May 2017 |
| Creators | Pattanavibool nee Vongvijitra, Rungnapar |
| Contributors | Aderkas, P. von |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
Page generated in 0.0027 seconds