This thesis reports the cell culture establishment and a somatic cell selection system optimized for the isolation of chlorsulfuron-resistant variants in asparagus (Asparagus officinalis L.). The development of this cell selection system benefited the isolation of chlorsulfuron-resistant variants from an elite asparagus genotype. A cell culture system, suitable for somatic cell selection, was established for asparagus genotype CRD 168. Friable callus was initiated from etiolated shoots in darkness and used to produce a high density of single cells in suspension. Cell density was estimated based on a linear relationship with settled cell volume. A mean plating efficiency of 0.19 % was recorded between 1-4x10⁵ cells/Petri dish. In vitro cell selection techniques were developed to identify mutant asparagus cells with resistance to a sulfonylurea herbicide, chlorsulfuron. A few key aspects were important to achieve this: a cell culture system for cell selection was initially established; a toxic concentration for the complete growth inhibition of the wild type asparagus cells was defined; rare, resistant cell colonies were isolated and characterized; and chlorsulfuron-resistant plants were regenerated. From about 50 million cells, 165 cell colonies were isolated in the presence of 8 nM chlorsulfuron. Characterization of these selected cell colonies yielded 24 escapes, 98 unstable variants, and 43 stable-resistant variants. Callus cultures from 34 of these stable variants retained resistance following 11 months growth in the absence of the selection agent. Plants were regenerated from 36 of these stable herbicide-resistant variants. Six of these chlorsulfuron-resistant variants were screened for their degree of resistance to chlorsulfuron, cross resistance to other acetohydroxyacid synthase (AHAS) inhibiting herbicides and AHAS enzyme activity. Cross resistance to imazamox was evident in four of the resistant variants, while lack of cross resistance to metsulfuron methyl was observed in all six resistant variants. A varying degree of resistance to chlorsulfuron was observed among the resistant variants. Both in the original and secondary callus, an uninhibited AHAS enzyme activity in all six resistant variants was recorded in the presence of high chlorsulfuron concentration (70-140 nM), compared to the total inhibition in the wild type. One chlorsulfuron-resistant variant, R-45, was used to compare the biochemical and physiological basis of resistance with the wild type. The AHAS enzyme activity in the tissue culture and greenhouse foliage of R-45 was significantly higher in the presence of up to 280 nM chlorsulfuron compared with the wild type. Chlorsulfuron retention was considerably higher due to the reduction of epicuticular wax deposits on the foliage of R-45, in comparison with the wild type. Consequently, the resistant line absorbed at least 1.6 fold more chlorsulfuron than the wild type plants. Therefore, foliar application of 15 g a.i./ha Glean (commercial formulation of chlorsulfuron) produced typical symptoms of chlorosis in R-45, similar to the wild type, in the greenhouse plants. Somatic cell selection was carried out using two elite asparagus genotypes, CRD 74 and Clone X. Of the 33 rare cell colonies isolated from Clone X, 22 unstable variants and 6 escapes were discarded. All five remaining resistant variants produced plants. One of the stable-resistant variants (Clone X-24) was evaluated for resistance to chlorsulfuron. Both in vitro shoot cultures and greenhouse-grown plants of Clone X-24 showed increased resistance to chlorsulfuron compared with the wild type. The AHAS enzyme activity in the foliar extracts also showed the presence of higher enzyme activity in Clone X-24.
| Identifer | oai:union.ndltd.org:ADTP/285828 |
| Date | January 1999 |
| Creators | Ganeshan, Dharshini |
| Publisher | Lincoln University |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://purl.org/net/lulib/thesisrights |
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