The major objective of this study was to produce synthetic genotypes of Brassica juncea from its parental species <i> B. rapa </i> and <i> B. nigra </i> via somatic and sexual hybridization. As prerequisites for somatic hybridization experiments, methods were developed to improve the culture of mesophyll and hypocotyl protoplasts of <i> B. nigra </i> and <i> B. rapa </i>, to obtain reliable plant regeneration from mesophyll protoplast cultures of <i> B. nigra </i>, and to fuse protoplasts of <i> B. nigra </i> and <i> B. rapa </i>. A modified Kao's medium (1977), was found suitable for the culture of mesophyll protoplasts of <i> B. nigra </i> and <i> B. rapa </i>. At a density of approximately $110\sp5$ protoplasts/ml within a culture plate insert surrounded by culture medium, mesophyll protoplast cultures of <i> B. nigra </i> accessions R890, R1819, R3392 and U1218 and <i> B. rapa </i> cvs. R500 and Wong Bok formed colonies. Genotypic differences in cell division and colony formation were observed. Hypocotyl protoplasts of <i> B. nigra </i> and <i> B. rapa </i> were successfully isolated from 6 day-old seedlings cultured in a modified Kao's medium (1977). With <i> B. nigra </i> accession R890 and <i> B. rapa </i> cv. R500, cell division and colony formation were optimal when hypocotyl protoplasts were cultured at a density of 0.5 to $1.010\sp5$ protoplasts/ml within a culture plate insert surrounded by a nurse culture of 4 to 6 day-old mesophyll protoplasts of <i> B. nigra </i>. Plant regeneration was obtained from mesophyll protoplast-derived calli of <i> B. nigra </i> accession R890 originally cultured in inserts; a shoot regeneration frequency of 8.1% was obtained on a medium containing the salts and vitamins of medium K3 (Nagy and Maliga 1976) with 3 g/l sucrose, 18.2 g/l mannitol, 2 mg/l ZR, 0.1 mg/l NAA, 10 g/l agarose, pH 5.6. For somatic hybridizatian studies, methods were developed to select out parental protoplasts using iodoacetic acid and to efficiently fuse protoplasts on the bottom of a petri dish using PEG. Twenty-nine plants were recovered from fusion experiments between mesophyll protoplasts of <i> B. nigra </i> accession R890 and hypocotyl protoplasts of <i> B. rapa </i> cv. Tobin. The somatic hybrid plants resembled natural <i> B. juncea </i>, had $2n=36$ chromosomes and had pollen viabilities ranging from 30 to 45%. Twenty-one plants, derived from one callus colony, possessed the mitochondrial and chloroplast genomes of <i> B. rapa </i>, as found in natural <i> B. juncea </i>. Eight plants, derived from another callus, had a novel cytoplasmic combination consisting of the mitochondrial genome of <i> B. rapa </i> and the chloroplast genome of <i> B. nigra </i>. Synthetic genotypes of <i> B. juncea </i> were also produced from reciprocal sexual crosses between <i> B. rapa </i> and <i> B. nigra </i>. Seventy-eight interspecific hybrid plants from the cross <i> B. rapa </i> x <i> B. nigra </i> and six hybrid plants from the reciprocal cross were identified by their morphology, pollen viability and chromosome number. The colchicine-induced allotetraploids resembled natural <i> B. juncea </i> in morphology, had 18 bivalents at metaphase I, and had between 35 and 70% pollen viability.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-10212004-000212 |
| Date | 01 January 1993 |
| Creators | Campbell, Craig Thomas |
| Contributors | Fowke, Larry C. |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-10212004-000212 |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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