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油菜(B.napus)花药培养中,经愈伤组织再分化获得植株,常受到花药体细胞组织的干扰,花粉植株形成的频率极低,本实验采用直接从花药诱导花粉胚状体长成小植株。用冬油菜杂交组合F_1为实验材料,以B_5为诱导培养基,取单核后期至双核初期的花药。接种前,花蕾经4—6℃预处理24小时,接种后花药在35℃高温下培养1天,接着在30℃培养6天,再转至25—27℃温度下持续培养,待胚状体形成后转移到MS培养基,诱导成小植株。细胞学观察:小孢子发育成为胚状体过程分三个阶段:(1)小孢子分裂阶段;(2)多细胞分裂发育阶段;(3)胚状体形成阶段。证实供体植株的基因型及生理状态、变温处理、培养基中蔗糖浓度和生长素用量是影响油菜花药培养诱导花粉胚频率的主要因素。
In B.napus anther culture, the plants were obtained by callus re-differentiation and were often disturbed by somatic tissue of anther. The frequency of pollen plants formation was extremely low. In this experiment, Plant. F_1, a winter rapeseed hybrid, was used as the experimental material and B_5 as the induction medium. Anther was collected from the late stage of mononuclear to the early stage of dual nucleus. Before inoculation, the buds were pretreated at 4-6 ° C for 24 hours. After inoculation, the anthers were cultured at 35 ° C for 1 day, then cultured at 30 ° C for 6 days, and then continued to culture at 25-27 ° C until the embryoid bodies After formation transferred to MS medium, induced into plantlets. Cytological observation: microspore development into embryoid body process is divided into three stages: (1) microspore division stage; (2) multicellular division development stage; (3) embryoid body formation stage. Confirming the genotypes and physiological status of donor plants, temperature-changing treatment, sucrose concentration in the medium and the amount of auxin were the main factors affecting the frequency of pollen embryos induced by anther culture.