Methodical improvements in microspore culture of Brassica napus L.

The utilisation of microspore in vitro culture and chromosome doubling for production of homozygous and doubled haploid plants is an important issue in modern breeding programs. Nevertheless, insufficient Colchicine induced diploidisation and direct embryo to plant conversion of microspore derived embryos still represent major drawbacks for an efficient application of the doubled haploid technology. Usually, most of the embryos undergo secondary embryogenesis which requires laborious, time- and cost-intensive multiple sub-culturing of the shoots. For that reason the following work, consisting of two studies, was conducted to further improve the doubled haploid technology in oilseed rape (Brassica napus L.). The aim of the first study was to enhance the Colchicine induced diploidisation frequency and to test the less toxic antimitotic herbicides Amiprophos-methyl (APM) and Pronamide with a higher affinity to plant tubulin without the reduction of direct embryo to plant conversion frequencies. The combination of antimitotic agents APM, Pronamide and Colchicine led to no efficient diploidisation frequency and consequently, no synergistic effect was detected. The 8 tested genotypes resulted in 40% to 64% diploid plantlets (means from all treatments). The diploidisation frequency of the treatments varied from 33% (3 µM APM) to 70% (25 µM Colchicine). A significant effect on the direct embryo to plant conversion rate was not detected and ranged from 14% to 23%. Different Colchicine concentrations (250, 150, 125, 25 µM) of four tested genotypes showed Colchicine induced diploidisation frequencies varying from 58% to 66%. The highest mean of 77% doubled haploid plantlets was achieved by the treatment with 250 µM Colchicine incubated for 48 hours. In practice antimitotic agents are usually dissolved in dimethyl sulphoxide (DMSO) with a final concentration from 0.03% to 3%. No significant effect of a low and a relatively high DMSO concentration in combination with a standard Colchicine (250 µM, 36 h) treatment was detected on diploidisation and direct embryo to plant conversion rate of four winter oilseed rape genotypes. In addition, the spontaneous and Colchicine induced diploidisation frequencies of 17 winter oilseed rape genotypes including cultivars and their F1-crosses were examined and the ability of their microspore derived embryos to convert directly to plantlets was evaluated. The spontaneous induced diploidisation frequency showed a wide range from 15% to 69% and the colchicine induced diploidisation frequency ranged from 40% to 83%. For all tested genotypes, the direct embryo to plant conversion rate widely varied from 2% to 35%. Regarding all experiments of the first study, the observed spontaneous and antimitotic induced diploidisation and the direct embryo to plant conversion frequency were genotype dependent. The aim of the second study was the improvement of the direct embryo to plant conversion frequency of microspore derived embryos and the reduction of secondary embryogenesis. In the first experiment the effect of ten shoot regeneration media supplemented with and without phytohormones (gibberellic acid, indole-3-butyric acid and 6-benzylaminopurine) and a 14-day cold treatment at 4 °C (Light Thermostat) of microspore derived embryos on direct embryo to plant conversion frequency was tested. The cold treatment with 4 °C was linked with eight hours and continuous light. For standard cultivation microspore derived embryos were exposed to 26 °C and 12 hours light. The five tested winter oilseed rape cultivars showed a range from 13% to 39% directly converted plantlets. The cultivation of microspore derived embryos on Gamborg B5 media supplemented with 0.1 mg/L gibberellic acid achieved the highest mean of 43%, while cultivation on B5 media supplemented with 0.1 mg/L indole-3-butyric acid and 0.2 mg/L 6-benzylaminopurine resulted in a mean of 11%, only. The two-week cold treatment significantly increased the frequency of direct embryo to plant conversion from 14% (under standard conditions) to 28%. In a following experiment, the effect of the four, previously most efficient, media and a 14-day cold treatment at 1.5 °C and at 4 °C (Light Thermostat) were tested on direct embryo to plant conversion frequency. The cold treatment at 1.5 °C was linked with eight hours of light and also with continuous darkness, while the cold treatment at 4 °C was linked with continuous light and continuous darkness. This experiment was conducted with 13 winter oilseed rape genotypes including cultivars and F1-hybrids and showed a wide range from 29% to 76% of direct embryo to plant conversion rate. In comparison to cultivation under standard conditions, the cold treatment at 1.5 °C and at 4 °C with and without light significantly increased the number of directly regenerated plantlets from 21% to 71%. For the tested culture media, the variation of direct embryo to plant conversion frequencies ranged from 50% (Murashige and Skoog) to 60% (Gamborg B5 with 0.1 mg/L gibberellic acid). Results of the study showed that although the ability of microspore derived embryos to convert directly to plantlets is mainly genotype dependent, a 14-days cold induction at 1.5 °C and at 4 °C significantly increased the direct embryo to plant conversion frequency. In conclusion, an efficient direct embryo to plant conversion of about 70% could be achieved for most of the tested genotypes by cold treatment at 1.5 °C without light. Therefore time- consuming in vitro-subcultivation could be considerably reduced resulting in an accelerated production of doubled haploid lines for application in practical oilseed rape breeding.