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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 (1): 7-8    
论文     
油菜小孢子培养以有效产生纯合两倍体植株
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: Since haploid embryoids were first obtained from microspore culture of Brassica napus there has been a rapid progress in the application of this technique in genetic studies and breeding utilization of rapeseed. The relative simplicity of the culture protocol and high-frequency recovery of doubled haploid plants makes microspore culture a potentially powerful technique for varietal development. Nevertheless, there are still some obstacles in the efficient use of this technique such as recalcitrant genotypes, secondary embryogenesis of embryoids, and the lack of efficient chromosome doubling. The present experiment is attempted to improve the production efficiency of doubled haploid rapeseed plants. 　　Several rapeseed accessions were used in the experiment. These donor plants were grown in the glasshouse with 16 h daylength and high nutritive status. A couple of days prior to microspore isolation temperature was kept down to 12　℃ during the day and preferably 10　℃ during the night. Buds at the late uninucleate stage were collected from racemes where one or two flowers had reached anthesis. 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. The microspore suspension was centrifuged at 750 r/min for 5 min and the microspore pellet was resuspended in 10 ml NLN-13 medium (13 % sucrose, full macro nutrients and without hormones, pH 6.0). 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 doubled sealed with Nescofilm, cultures were incubated at 30　℃ for 7 days and then moved to 24　℃ in the dark. As soon as embryos were visible to naked eyes, cultures were transferred to a slow rotary shaker (45 r/min). At the late torpedo stage, 15 embryos were transferred to solid MS medium (2% sucrose, half-strength macro nutrients and 0.1 mg/L GA3, solidified with 0.9% agar, pH 5.8) 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. Recalcitrant embryos could be trimmed by cutting to induce germination. 　　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 levels was made on leaf tissue samples about 3 weeks after planting in soil. Leaf tissue was placed in a 6 cm petri dish in the fluorochrome DAPI (4′,6-diamidino-2-phenylindole) and homogenized by chopping with a sharp razor blade. The suspension of nuclei and cellular debris was filtered by passing through a 40 μm nylon gauze and the filtrate was immediately analyzed with the flow-cytometer. The channel for the DNA contents of the diploid peak was identified by using leaf tissue from normal diploid rapeseed as a standard. The DNA distribution curves were automatically analyzed by the flow-cytometer and recorded by a printer. The DNA peaks were compared to the standard peak and assigned to the ploidy level of haploid, diploid, tetraploid, etc. 　　35%-45% of spontaneous doubling frequency was obtained from the majority of rapeseed genotypes with the range from 10% to 60%. Diploid plantlets were transferred to the plant nursery for seed increase while haploid plantlets were kept dry one or two days and then washed free of soil. The roots were treated with 125 mg/L colchicine for 20 h and then washed once in water. After replanting in soil-perlite mixture the plants were handled
出版日期: 2000-01-10
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引用本文:

周伟军　HAGBERG Per. 油菜小孢子培养以有效产生纯合两倍体植株[J]. 浙江大学学报(农业与生命科学版), 2000, 26(1): 7-8.

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https://www.zjujournals.com/agr/CN/Y2000/V26/I1/7

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