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美国花旗松组织培养及再生系统建立的研究

Study on the Technique of Tissue Culture and Regeneration of Douglas-fir

【作者】 陆燕元

【导师】 樊军锋;

【作者基本信息】 西北农林科技大学 , 林木遗传育种, 2005, 硕士

【摘要】 美国花旗松[Pseudotsuga menziesii (Mirb.) Franco]是世界著名用材兼观赏树种。它抗逆性强,材质优良,树姿优美,是世界有名的“圣诞树”,也是美国、加拿大等国主要的造林树种。目前已经在我国的许多地区引种成功,但由于结实间期长,种子产量低,远远不能满足实际需要,而且采用扦插技术繁殖时生根特别困难,采用的材料一般要是小于十年生的枝条,这都限制了花旗松的的进一步推广繁殖。因此,进行美国花旗松离体培养和植株再生研究在理论和实践上均有重要意义。它不仅在短期内扩大繁殖现有的优质材料,为生产提供大量的优质苗木,而且为花旗松农杆菌介导的遗传转化体系、改良松树遗传品质提供了必要前提和条件。 本文以美国花旗松成熟胚和实生苗为材料,研究其组织培养技术及再生体系的建立。其主要研究结果如下: (1)以美国花旗松成熟合子胚为外植体进行愈伤组织的诱导时,基本培养基对其有一定的影响。高盐浓度的培养基有利于愈伤组织的诱导;降低盐浓度,则会减少愈伤组织的形成量。激素在愈伤组织的诱导中起关键作用。单独使用细胞分裂素6-BA  2.0mg/L 就可以诱导出质量较好的愈伤组织,配合使用一定浓度的NAA,愈伤组织形成量增加,NAA 浓度过高,在继代中褐化严重。改良DCR+2.0mg/L6-BA+0.01 mg/L NAA 为诱导美国花旗松愈伤组织最佳的培养基组合。蔗糖对愈伤组织的诱导也有很大的作用。2%-3%浓度的蔗糖诱导效果最好。 (2)以美国花旗松成熟合子胚为外植体进行不定芽的诱导时,基本培养基和激素均有重要的影响。供试的6 种基本培养基中,改良DCR最好,WPM、SH 次之,GD 较差,MS 和N6 则不适用。单独添加2.0mg/L细胞分裂素6-BA 对不定芽的诱导效果最好。较为适宜的培养基组合为改良DCR+2.0mg/L 6-BA,诱导率可达到74%,平均增殖系数为6,最大的增殖系数可达到11。(3)外植体的处理方式对不定芽诱导有很大的影响。预先用高浓度(50mg/L)的细胞分裂素6-BA 进行浸泡处理,胚的萌发率及诱导率都较低,污染率也很高,但通过此方式却可以获得少量不经愈伤组织而直接分化出的不定芽。而直接接种到诱导芽培养基的成熟胚只能从愈伤组织中分化出不定芽。 (4)以带子叶顶芽为外植体建立美国花旗松组织培养的再生体系相对较难。在最适合带子叶顶芽不定芽诱导的培养基组合改良DCR+3.0mg/L 6-BA 中,带子叶顶芽的无菌苗和实生苗不定芽的诱导率都较低。而在两者中以实生苗最低,诱导率只有22.6%,分化系数为3.6, 无菌苗可达到42.8%,分化系数为3.8。

【Abstract】 Douglas-fir[Pseudotsuga menziesii] is one of the most important and valuable timber trees in the world. It is a major component of the forests in America and Canada. Presently, it has been successfully introduced in many reigions in China. Because the seed production is low,and the seeding time is long , the seed supply are far away from the needs of afforestation. Cuttings as a regeneration procedure has demonstrated that reliable rooting of cuttings was limited to the material trees less than 10 years old. All these limit douglas-fir to spread. So the study of Douglas-fir in vitro and regeneration is important in theory and practise. It not only can supply a lot of seedlings which has good wood quality at short time, it also tentative explore the effective way of Douglas-fir for plant breeding providing some theorial bases. This article maked mature embryo and seedlings of Douglas-fir as explant, researched the system of the rapid proliferation in Douglas-fir by the way of tissue culture. The results were as following: (1) When callus development was initiated with the mature embryo of Douglas-fir, the basal medium had some influence. Medium of high salt concentration favoured development of callus. Decreasing the salt concentration of medium would reduce the callus. Hormone in this prosure was a key factor. Only supplement 6-BA was enough on induction adventitous buds. Supplemented with some concentration of NAA can increase callus number, however, when the concentration of NNA was too high, the browning rate of callus would be very high in subculture. The combination medium of modified DCR + 2.0 mg/L 6-BA + 0.01 mg/L NAA was optimal. Meanwhile, sucrose also had some influence on callus formation. 2%-3% of sucrose was the most optimal concentration. (2) During the prosure of adventitious bud formation from mature embryos, basal medium and hormone also made important role. In the six basal mediums, modified DCR was the best, WPM and SH were better than GD, MS and N6 were the worse. Only supplemented with 2.0mg/L 6-BA in adventitious buds formation, the rate of induction was highest. The optimal combination of medium was modified DCR+2.0 mg/L6-BA, on which 74% explants was induced , average inductive rate of propation was 6, and the highest rate of propagation was 11. (3) Different treatment of explants also had important influence. Soaked embryos in high concentration hormone before culture, the rate of germination and induction both were low and contamination rate was high, however, some adventitious buds has been gotwhich induced directly from explants. Cultured directly in inductive medium, adventitious buds only induced from callus. (4) It was showed by the experiment that selecting the sprout carrying cotyledons as explants was more difficult to built the regeneration system. As if in the optimal combination of medium modified DCR+3.0 mg/L6-BA, the inductive rate of both seedling which came from tests and soil were low. In the two kinds of seedlings, the later was worse,which inductive rate was only 22.6%, and the buds proliferation coefficient was 3.6; the other, the two datas were respectively 42.8% and 3.8. (5) Different treatment way of adventitious buds was important in tufted buds growth. Soaked adventitious buds in 18.26 mg/L NAA and 20.32 mg/L IBA (0.1mmol/L) before cultured in medium would promote the elongation of tufted buds. Especially, treated in the mix hormone for 3 hours, the growth of buds reached 1.2 cm in a month. In 1/2SH medium without hormone,average of growth of adventitious buds was 0.7cm in a month. Active charcoal was advantageous to continue development for tufted buds. GA3 was harmful to it. (6) In the experiment of Douglas-fir rooting, we obtained such a conclusion: rooting in out-test was better than in in-test. In out-test, the rooting rate of Douglas-fir was 12.7%, but even in the optimal combination of medium 1/2SH+NAA0.01 mg/L+IBA1.0mg/L, the rooting rate was only 5.2%.

  • 【分类号】S791.161
  • 【被引频次】7
  • 【下载频次】308
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