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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2000, Vol. 26 Issue (2): 125-126    
论文     
油菜小孢子秋水仙素直接处理增加纯合两倍体植株频率
周伟军　HAGBERG Per
周伟军（浙江大学 农业与生物技术学院，浙江 杭州310029） HAGBERG Per（Nilsson-Ehle Laboratory, Svalof Weibull AB, Svalov 26881, Sweden）
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Abstract: Techniques for microspore culture of Brassica napus have been improved rapidly, and embryogenesis has been achieved in a wide range of genotypes. Plants regenerated from microspore-derived embryoids can be haploid, diploid or polyploid. From rapeseed microspore culture it is reported that 70%-90% of regenerated plants are haploid. The usual methods of chromosome doubling involve soaking roots (most common) or whole plants in a colchicine solution, or culturing plantlets in colchicine-containing medium in the greenhouse. Other alternatives are injecting colchicine into the secondary buds or applying colchicine-soaked cotton plugs to axillary buds. 　　All these methods have shortcomings in an intensive plant breeding programme. They are relatively labour intensive and time consuming, and also generate large volumes of colchicine, a problem which requires specialized handling and disposal. Meanwhile, these treatments substantially retards plant growth and often only a portion of the plant becomes diploidized. Consequently, the doubled plants can not produce enough seeds for field trials or other analysis and an additional growth cycle is necessary for seed increase. 　　Colchicine can be practically applied at any stage during the microspore culture process, from isolated microspores to the regenerated plants. Colchicine added in vitro to the induction medium of freshly isolated rapeseed microspores in the first day of culture reportedly produced an improved embryogenesis and no negative effect on their further development. Therefore, the present experiment was performed to study the efficiency of three chromosome doubling methods in rapeseed, i.e., colchicine treatments of microspore-derived plants, microspore-derived embryos, and direct colchicine treatment of isolated microspores. 　　Genetic materials used for microspore culture included the Brassica napus accessions “5320”, “5389” and “5392”. Donor plants were grown in a growth chamber at 12 ℃/10 ℃ (day/night) with 16 h daylength. Buds at the late uninucleate stage were collected from racemes of rape plant. After surface sterilized in Gevonium for 18 min and washed 3 times in sterile water, 18 buds were crushed gently in 10 ml B5 medium and filtered through a 40 μm nylon filter. A 0.2% stock solution of colchicine in the induction medium (NLN-13 medium) was prepared and filter-sterilized using a 0.2 μm bacterial filter. Three methods of chromosome doubling to produce doubled haploid plants from rapeseed microspore cultures were compared: Colchicine treatments of microspore-derived plants (plant roots treated with 125 mg/L colchicine for 20 h), microspore-derived embryos before transferring to the solid MS medium, and direct colchicine treatment of freshly isolated microspores. 　　The microspore suspension was centrifuged at 750 r/min for 5 min and the microspore pellet was resuspended in 10 mL NLN-13 medium. 0.5 mL of suspension was portioned into each one 6 cm petri dish. NLN-13 medium was added to just cover the bottom of each dish and then added a drop of charcoal suspended in NLN-13 with agarose. After sealed with Nescofilm, cultures were incubated at 30 ℃ for a total of 7 days and then moved to 24 ℃ in the dark with a slow rotary shaker (45 r/min). At the late torpedo stage, 15 embryos were transferred to solid MS medium in each plastic growth container. After an initial period of 10 days at 2　℃ cultures were incubated in a growth chamber with 24　℃ and 16 h daylength and low light intensity. When shoots developed they were cut free of any callus or meristematic tissue and transferred to larger growth vessels with solid MS medium where rooting takes place. Plantlets were transferred to soil-perlite mixture and kept for 2 weeks in a nursing room with a temperature of 24　℃, 16 h daylength, low light intensity and high relative humidity. Gradual adaptation to glasshouse conditions followed. Flow-cytometer (Partec Ploidy Analyzer, Partec GmbH, Germany) analysis of ploidy leve
出版日期: 2000-03-13
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引用本文:

周伟军　HAGBERG Per. 油菜小孢子秋水仙素直接处理增加纯合两倍体植株频率[J]. 浙江大学学报(农业与生命科学版), 2000, 26(2): 125-126.

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https://www.zjujournals.com/agr/CN/Y2000/V26/I2/125

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