花楸体细胞胚高频诱导及萌发促进的研究
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摘要
花楸(Sorbus pohuashanensis),蔷薇科花楸属落叶小乔木,是我国北方特有的园林观赏性树种。花楸体胚发生及植株再生的研究对花楸优良无性系的大量快繁具有重要意义。本文针对花楸体胚诱导率和萌发率低的问题,通过对外植体进行预处理的方法来提高花楸体胚的诱导率,同时采用扫描及透射电子显微镜观察的方法跟踪观察花楸体胚发生过程中细胞的变化,并从细胞的角度分析了体胚高频发生的原因;在此基础上进行次生胚的增殖和成熟的研究,找出促进花楸体胚萌发的重要影响因素,为花楸体细胞胚胎发生及植株再生体系的建立提供科学的理论依据。
(1)以花楸未成熟合子胚为外植体作为体胚诱导材料,选择未经任何预处理的外植体作为对照;使用1 mol·L~(-1)的甘露醇对外植体渗透预处理24 h后,体胚的诱导率得到明显的提高,并且体胚直接发生于外植体表面,说明甘露醇对外植体的渗透预处理有利于花楸体胚的发生。同时,通过扫描及透射电子显微镜对外植体进行观察发现,与对照相比经过预处理的外植体细胞内部的变化从培养初期就有巨大差异。子叶部位较对照有着明显的变化,其边缘的表面细胞不规则突起且细胞排列疏松,细胞内部间隙变大且细胞壁明显加厚,可能这种细胞上的变化有利于多细胞原胚的发生,为体胚的高频率发生提供了保证。
(2)对已获得的花楸体胚及体胚团块进行增殖与成熟培养,在光照条件下,体胚在添加生长素萘乙酸的MS培养基上,接种密度为每皿30个体胚时,体胚的增殖效果最佳;花楸体胚的在黑暗条件下,以MS培养基为基本培养基并附加5%的蔗糖和200mg·L~(-1)水解酪蛋白,添加附加物聚乙二醇能够有效的促使体胚成熟,对体胚进行此成熟处理后体胚的成熟率可达59%。成熟的花楸体胚在形态上表现为乳白色或浅黄色不透明结实的子叶形体胚,长度大约4-5 mm。
(3)选择成熟较好的体胚进行萌发试验,体胚经过成熟培养35天后,将其接种于添加少量生长素和0.1%活性炭的MS萌发培养基上培养,体胚的萌发率较高,并且体胚的两片真叶发育正常且部分长出根。
总结以上分析可以得出结论,有效的甘露醇渗透预处理可以促使花楸体胚的高频率发生;通过人为控制的方法可以掌握体胚增殖与成熟的基本培养条件。
Sorbus pohuashanesis Hedl.is an important ornamental tree species in north China, Somatic embryogenesis and plant regeneration plays an important role in establishing fast propagation systems for improved materials with special traits.High efficiency induction and germination promotion of somatic embryos of Sorbus pohuashanesis were studied here.The somatic embryo induction process were observed simultaneously by useing scanning electron microscopy(SEM) and transmission electron microscopy(TEM).The results are as follows:
(1) Somatic embryogenesis of S.pohuashanesis on cotyledon explants treated by 1 mol·L~(-1) mannito pretreatment the for 24 hours was improved evidently,and the Somatic embryos regenerated directlyfrom surface cell of the explants.SEM and TEM observation revealed that epidermal cell lineage of cotyledon explants pretreatmented with mannitol turned rapidly to irregular,cell wall became more thick and cell division planes was random.This indicates that pretreatment might strongly disturb the cell-to-cell relationship and produced a favorable status for somatic embryogenesis.
(2) MS + 0.01mg·L~(-1) rhodofix and incubated 30 somatic embryo explants per dish under illumination was most favorable to proliferation of secondary somatic embryogenesis,the proliferation rate could be reached high-level.Cultured in dark was propitious to somatic embryo maturation,on the optimum medium of MS + 200 mg·L~(-1) CH + 5%sucrose + 5 g·L~(-1) PEG6000,the maturation rate could be achieve to 59%.Appearance of mature somatic embryos would be described as cotyledons thick,milkly or pale yellow and about 4-5 mm long in diameter.
(3) After maturation cultured for 35 days,the somatic embryos germinated well on MS+ auxin + 0.1%active carbon,germination rate could be reached 50%.
The above results showed that effective mannitol pretreatment can promote high efficiency induction of somatic embryos of Sorbus pohuashanensis and the basic conditions for proliferation and maturation of the somatic embryos can be controled artificially.
(1)以花楸未成熟合子胚为外植体作为体胚诱导材料,选择未经任何预处理的外植体作为对照;使用1 mol·L~(-1)的甘露醇对外植体渗透预处理24 h后,体胚的诱导率得到明显的提高,并且体胚直接发生于外植体表面,说明甘露醇对外植体的渗透预处理有利于花楸体胚的发生。同时,通过扫描及透射电子显微镜对外植体进行观察发现,与对照相比经过预处理的外植体细胞内部的变化从培养初期就有巨大差异。子叶部位较对照有着明显的变化,其边缘的表面细胞不规则突起且细胞排列疏松,细胞内部间隙变大且细胞壁明显加厚,可能这种细胞上的变化有利于多细胞原胚的发生,为体胚的高频率发生提供了保证。
(2)对已获得的花楸体胚及体胚团块进行增殖与成熟培养,在光照条件下,体胚在添加生长素萘乙酸的MS培养基上,接种密度为每皿30个体胚时,体胚的增殖效果最佳;花楸体胚的在黑暗条件下,以MS培养基为基本培养基并附加5%的蔗糖和200mg·L~(-1)水解酪蛋白,添加附加物聚乙二醇能够有效的促使体胚成熟,对体胚进行此成熟处理后体胚的成熟率可达59%。成熟的花楸体胚在形态上表现为乳白色或浅黄色不透明结实的子叶形体胚,长度大约4-5 mm。
(3)选择成熟较好的体胚进行萌发试验,体胚经过成熟培养35天后,将其接种于添加少量生长素和0.1%活性炭的MS萌发培养基上培养,体胚的萌发率较高,并且体胚的两片真叶发育正常且部分长出根。
总结以上分析可以得出结论,有效的甘露醇渗透预处理可以促使花楸体胚的高频率发生;通过人为控制的方法可以掌握体胚增殖与成熟的基本培养条件。
Sorbus pohuashanesis Hedl.is an important ornamental tree species in north China, Somatic embryogenesis and plant regeneration plays an important role in establishing fast propagation systems for improved materials with special traits.High efficiency induction and germination promotion of somatic embryos of Sorbus pohuashanesis were studied here.The somatic embryo induction process were observed simultaneously by useing scanning electron microscopy(SEM) and transmission electron microscopy(TEM).The results are as follows:
(1) Somatic embryogenesis of S.pohuashanesis on cotyledon explants treated by 1 mol·L~(-1) mannito pretreatment the for 24 hours was improved evidently,and the Somatic embryos regenerated directlyfrom surface cell of the explants.SEM and TEM observation revealed that epidermal cell lineage of cotyledon explants pretreatmented with mannitol turned rapidly to irregular,cell wall became more thick and cell division planes was random.This indicates that pretreatment might strongly disturb the cell-to-cell relationship and produced a favorable status for somatic embryogenesis.
(2) MS + 0.01mg·L~(-1) rhodofix and incubated 30 somatic embryo explants per dish under illumination was most favorable to proliferation of secondary somatic embryogenesis,the proliferation rate could be reached high-level.Cultured in dark was propitious to somatic embryo maturation,on the optimum medium of MS + 200 mg·L~(-1) CH + 5%sucrose + 5 g·L~(-1) PEG6000,the maturation rate could be achieve to 59%.Appearance of mature somatic embryos would be described as cotyledons thick,milkly or pale yellow and about 4-5 mm long in diameter.
(3) After maturation cultured for 35 days,the somatic embryos germinated well on MS+ auxin + 0.1%active carbon,germination rate could be reached 50%.
The above results showed that effective mannitol pretreatment can promote high efficiency induction of somatic embryos of Sorbus pohuashanensis and the basic conditions for proliferation and maturation of the somatic embryos can be controled artificially.
引文
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