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以 2个对小孢子培养反应较好的甘蓝型冬油菜F1杂种为供体材料 ,研究了 3种不同时期秋水仙碱处理对小孢子胚胎发生、成苗与加倍率的影响。对分离小孢子直接进行 15h以及 0、5 0、5 0 0和 10 0 0mg/L秋水仙碱处理 ,在液体NLN培养基中胚胎发生好 ,培养 5周后获得大量正常胚体。将这些发育优异的胚体移入固体MS培养基、经过 10d低温 (2℃ )诱发 ,常温 (2 4℃ )下培养能很好地萌发 ,并迅速长成完全正常的再生植株。 5 0 0mg/L秋水仙碱处理 15h获得了 83%~ 92 %的高染色体加倍率 ,并且嵌合体和多倍体数很少。对正常胚体在移入固体培养基前用 0、2 5、5 0、2 5 0、5 0 0和 10 0 0mg/L秋水仙碱 15和 30h进行染色体加倍处理 ,或对再生植株在幼苗期进行 12 5mg/L秋水仙碱 2 0h浸根处理 ,其加倍效果均不及采用分离小孢子直接进行秋水仙碱处理 ,且其嵌合体的发生频率也较高 ;后者还产生大量待特殊处置的秋水仙碱废液 ,并推迟再生株的生育进程。因此 ,利用分离小孢子直接进行秋水仙碱加倍染色体更为安全、快速和有效
Two F1 hybrids of Brassica campestris with better response to microspore culture were used as donor materials to study the effects of colchicine treatment on microspore embryogenesis, seedling emergence and doubling rate in three different periods. Microspores were directly isolated for 15h and 0, 50, 0, 0 and 100 mg / L colchicine. The embryos were well cultured in liquid NLN medium and a large number of normal embryos were obtained after 5 weeks of culture. After these embryos were transferred into solid MS medium for 10 days, they were germinated under normal temperature (2 4 ℃) and grew into completely normal regenerated plants after 10 days hypothermia (2 ℃). Colchicine treatment with 500 mg / L colchicine for 15 h resulted in a high chromosome doubling rate of 83% -92%, with few chimeras and polyploidy. The normal embryo bodies were treated with colchicine at 0, 25, 50, 25 0, 500 and 100 mg / L colchicine for 15 and 30 h prior to transplanting into solid medium, When 12 5 mg / L colchicine was immersed in root for 20 h, the doubling effect was not as good as that of using colchicine to separate colchicine, and the frequency of its chimera was higher. The latter also produced a large amount of Colchicine waste, and postponed the reproductive process of fertility. Therefore, it is safer, faster and more effective to directly colchicine doubling chromosomes by separating microspores