Improvement on the microspore culture methodology for Brassica rapa canola
Stadnyk, Kimberly Dawn
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The objective of this research is to improve the microspore culture methodology based on embryo production levels for B. rapa. An efficient isolated microspore culture methodology for Brassica rapa canola could be used by breeding programs to produce high, consistent embryo yields. The project was divided into several experiments looking at modifications to the culture media, microspore pretreatments, microspore liberation techniques and donor plant pretreatments. Four B. rapa genotypes were selected for the experiments: Reward, AC Parkland, AC-1 and AC-2. The standard protocol used in this research followed Baillie et al. 1992. Plant Cell Reports 11:234-237. Experiment 1 was an evaluation of the effect of sucrose levels in the microspore culture media on embryo production of B. rapa... Experiment 2 was an evaluation of the effect of pH levels of the microspore culture media on embryo production of B. rapa... Experiment 3 was an evaluation of the effect of microspore pretreatment in the form of a cold shock on embryo production in B. rapa... Experiment 4 was an investigation of a large-scale microspore culture technique and its effects on green embryo production in B. rapa... Experiment 5 was an evaluation of the effect of donor plant pretreatment on embryo production in B. rapa... The two growth retardants A-Rest [a-cyclopropyl-a-(4-methoxyphenyl)-5-pyrimidin-5-yl)benzyl alcohol] and Cycocel (2-chloroethyl trimethyl-ammonium-chloride) were used to investigate if a growth retardant could replace the requirement of low temperature donor plant growing conditions in the microspore culture of B. rapa. The intent was to mimic retarded development achieved under low temperature growth conditions with the growth retardants. Cytological observations suggest that A-Rest has a negative effect on the percent of microspores in the mid-late uninucleate stage of development for Reward while AC-2 is unaffected. Cycocel seems to negatively affect the percent of microspores in the mid-late uninucleate stage of development for AC-2 while it has no effect on Reward. Successful application of the isolated microspore culture technique is determined by the induction of response in many genotypes. The practical approach is to determine conditions and treatments which result in the best average embryo response across all of the genotypes being utilized rather than striving for optimal conditions for each genotype.