Methodical improvements in microspore culture of Brassica napus L.
Methodical improvements in microspore culture of Brassica napus L.
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甘蓝型油菜小孢子培养的方法改进。
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发表时间：
2013
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通讯作者：
Sarah Klutschewski
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作者：
Sarah Klutschewski
利用小孢子体外培养和染色体加倍技术生产纯合和加倍的单倍体植株是现代育种中的一个重要问题。然而，秋水仙素不足诱导的二倍体和小孢子胚向植株的直接转化仍然是有效应用加倍单倍体技术的主要缺陷。通常，大多数胚胎经历二次胚胎发生，这需要费力、耗时和成本高的芽多次继代培养。为此，为进一步完善甘蓝型油菜(Brassica napus L.)加倍单倍体技术，开展了以下两项研究工作。
第一项研究的目的是提高秋水仙素诱导的二倍体频率，并在不降低胚胎到植物的直接转化频率的情况下，测试毒性较低的与植物微管蛋白亲和力较高的抗有丝分裂除草剂氨虫草胺(APM)和普罗尼胺(Pronamide)。APM、普罗尼胺和秋水仙碱联合使用时，没有有效的二倍体率，因此没有检测到协同作用。
8个供试基因型的二倍体植株成苗率为40%至%(平均为所有处理)。这些处理的二倍化频率从33%(3uM APM)到70%(25uM秋水仙素)不等。对胚苗的直接转化率影响不大，在14%~23%之间。不同浓度的秋水仙碱(250、150、125、25µM)对四种供试基因的诱导倍增率从58%到66%不等。250µM秋水仙素处理48h，单倍体成苗率最高，为77%。在实践中，抗有丝分裂药物通常溶解在二甲基亚砜(DMSO)中，最终浓度在0.03%到3%之间。低浓度和较高浓度的DMSO与标准秋水仙素(250µM，36h)处理对4个油菜品种的二倍体和胚植株转化率没有显著影响。此外，还检测了17个油菜品种及其F1组合的自发和秋水仙素诱导的二倍体频率，并评价了它们的小孢子胚胎直接转化成植株的能力。自发诱导的二倍体率为15%~69%，秋水仙碱诱导的二倍体率为40%~83%。在所有被测试的基因型中，胚胎到植株的直接转化率在2%到35%之间变化很大。在第一个研究的所有实验中，观察到的自发和反有丝分裂诱导的二倍体以及胚胎到植物的直接转化频率是由基因型决定的。第二项研究的目的是提高小孢子胚直接转化为植株的频率，减少二次胚胎发生。在第一个实验中，研究了10种附加和不添加植物激素(赤霉酸、吲哚-3-丁酸和6-苄基氨基嘌呤)的不定芽再生培养基和4℃(光温)冷处理14天对小孢子胚芽直接转化植株的影响。冷处理温度为4℃，处理时间为8h，光照连续。在标准培养条件下，将小孢子胚胎置于26℃、12h的光照下。供试的5个冬季油菜品种的直接转化植株的比例从13%到39%不等。在含L赤霉酸0.1 mg/L的Gamborg B5培养基上培养的小孢子胚得率最高，平均为43%，而在添加0.1 mg/L吲哚-3-丁酸和0.2 mg/L 6-苯氨基嘌呤的B5培养基上培养的小孢子胚得率最高，仅为11%。两周的冷处理显著提高了胚向植株的直接转化率，从14%(标准条件下)提高到28%。
在接下来的实验中，测试了四种以前最有效的培养基和在1.5℃和4℃(光恒温器)下14天的冷处理对胚胎到植株的直接转化频率的影响。1.5℃的冷处理与8小时的光照和连续的黑暗有关，而4℃的冷处理与连续的光照和连续的黑暗有关。本试验以13个冬用油菜品种和F_1代为材料，获得了29%~76%的胚苗直接转化率。与标准培养条件相比，1.5℃和4℃有光和无光的低温处理显著提高了直接再生植株的数量，从21%增加到71%。在所试验的培养基中，胚芽直接转化成植株的频率变化范围从50%(Murashige和Skoog)到60%(Gamborg B5+0.1 mg/L赤霉酸)。
研究结果表明，尽管小孢子胚胎直接转化成植株的能力主要取决于基因型，但在1.5℃和4℃下进行14天的低温诱导显著提高了胚胎到植株的直接转化频率。
综上所述，在1.5℃的无光照条件下，大多数供试品种都能获得70%左右的直接胚苗转化效率。因此，可以大大减少耗时的试管继代培养，从而加速产生可应用于实际油菜育种的加倍单倍体品系。
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.