摘要
本研究以墨西哥落羽杉为对象,进行了体细胞胚胎发生和植株再生、器官发生和植株再生、试管无性系快速繁殖等方面的研究,主要结果如下:
1.以墨西哥落羽杉(Taxodium mucronatum)种子萌发后的下胚轴和子叶为外植体,进行墨西哥落羽杉直接体细胞胚胎发生的研究。结果表明:子叶外植体未能诱导出体细胞胚,与子叶相比,下胚轴更有利于体细胞胚胎的发生,但下胚轴培养时间的长短也直接影响体细胞胚胎发生频率。取种子萌发、子叶展开一周后的下胚轴在DCR+BA2.0mg/l+IBA0.1mg/l的培养基上直接体细胞胚发生频率可达24.5%。诱导出的体细胞胚及时转入不添加任何激素的DCR基本培养基中以促进其萌发。
2.以墨西哥落羽杉种子萌发后的下胚轴和子叶为外植体,进行器官发生的研究。结果表明:子叶未能诱导产生不定芽。下胚轴在DCR+BA2.0mg/l+IBA0.1mg/l+TDZ0.01mg/l的培养基上不定芽诱导频率为58.5%,低浓度的TDZ与BA配合使用有利于下胚轴不定芽的诱导与分化。诱导出的不定芽继代在不添加任何激素的DCR基本培养基中伸长培养,在不添加任何激素的1/2DCR基本培养基中进行生根诱导,生根诱导率可达70%以上。
3.通过幼苗和腋芽两种不同外植体来建立墨西哥落羽杉试管无性系。结果表明:幼苗在DCR+BA0.5mg/l+IBA0.2mg/l培养基上诱导分化效果最好,分化频率80%以上,平均每个外植体产芽2-5个。诱导出针叶基潜伏芽后,转入DCR+500mg/lGln中伸长生长。Gln能够促进针叶基潜伏芽的伸长生长。试管苗在3/4DCR+0.05%AC培养基中生根效果最好,生根率可达78%。选择节间较长,生长旺盛,茎干较粗壮的小苗诱导生根过程往往容易完成。根长达2-4cm时,开瓶炼苗3-7天后即可进行移栽。
4.DCR+IBA0.05mg/l+BA0.025mg/l培养基最有利于茎段腋芽的抽梢与伸长生长。腋芽针叶基潜伏芽的诱导以DCR+BA0.3mg/l+NAA0.1mg/l+Gln500mg/l培养基为最佳,加入Gln可明显提高腋芽针叶基潜伏芽的诱导率。TDZ不适宜墨西哥落羽杉腋芽针叶基潜伏芽的诱导。针叶基潜伏芽在DCR+0.1%AC的培养基中伸长效果最好,继代2-3次后即可剪切较长的芽用于生根。以腋芽针叶基潜伏芽增殖方式建立起来的试管无性系诱导生根困难,生根频率较低。
上述研究为发展墨西哥落羽杉器官发生、体细胞胚胎发生诱导技术、以及墨西哥落羽杉无性系的快速繁殖,进而开展遗传转化研究奠定了良好的基础。
Somatic embryogenesis, direct organogenesis and plantlet clone for Taxodium mucronatum were conducted in this research.. The main results as follows:
1. Hypocotyl and cotyledon developed from the seeds of Taxodium mucronatum were cultured as the initiation explants for direct inducement of somatic embryos. Comparing with cotyledon, hypocotyl was better for us to induce somatic embryos, and the hypocotyl culture times directly effect on inducement frequency of somatic embryos also. The hypocotyl direct inducement frequency was 24.5% on the DCR medium with 2.0mg/l BA+0.1mg/lIBA. It is necessary for the development of somatic embryos by transferring the culture materials on hormone-free DCR medium.
2. Hypocotyl and cotyledon developed from the seeds of Taxodium mucronatum were also cultured as the initiation explants for direct organogenesis. It was not able to induce adventitious buds by the explant of cotyledon. With hypocotyl explants, the highest inducement frequency of adventitious buds was 58.5% on the DCR medium with 2.0mg/l BA, 0.1mg/1 IBA and 0.01mg/1 TDZ. The medium with low concentration of TDZ and BA was good to inducement and differentiation of adventitious buds of hypocotyl. Adventitious buds cultured on hormone-free DCR medium for elongation, subculture every four or five weeks, and cut the buds for rooting inducement after subculture two or three times. On hormone-free 1/2DCR medium the rooting frequency could be above 70%.
3. There are two kinds of explants to be used for plantlet clone of Taxodium mucronatum. The first one was the young seedling developed from the seeds. The results were shown that: dormant buds induced are sensitive to the different hormone concentration. Cultured young seedlings on DCR medium supplemented with 0.5 mg/1 BA and 0.2mg/l IBA was the best way of inducement, and with inducement frequency higher than 80%, mean number of dormant buds was around 2-5. The medium should be wiped off hormone when dormant buds induced. For elongation of the dormant buds, the suitable culture medium was DCR+Gln500mg/l. It can advance elongation of dormant buds when DCR basal medium supplemented with 500mg/lGln. The medium of 3/4DCR+0.05%AC is used for rooting, and the highest rooting rate is 78%. Using hormone to induce roots could produce mass of callus on the end top of cuttings and it was not benefit to the plantlets survive after transplant. In addition, it was easily for the plantlets with long internode and vigorous stem to induce root system. When the roots developed to 2-4cm long, the plant could be transplanted after 3-7 days acclimation
4. The second kinds of explants for plantlet clone was axillary buds developed from stem. The best inducement and elongation medium for axillary buds was DCR medium supplemented with 0.05mg/l IBA and 0.025mg/l BA, and inducement frequency up to 100%. Cutting down longer axillary buds from stem was good to other smaller axillary buds developed, and more new axillary buds produced from stem. The proper medium for inducing dormant buds from axillary buds was DCR medium supplemented with 0.3mg/l BA, 0.1 mg/1 NAA and 500mg/l Gln. The frequency of inducement was significant increased by adding Gin. TDZ was not suitable for dormant buds inducing from axillary buds. The best medium for elongating induced dormant buds was DCR+0.1%AC, subculturing 2-3 times, cutting down for rooting. The best medium for rooting in this research is 3/4DCR medium supplemented with 0.1 mg/1 IBA and 0.05mg/l NAA, however, the rooting rate was only 25%.
The frameworks for somatic embryogenesis, organogenesis, and micropropagation as mentioned above were established, and then the genie transformation of Taxodium mucronatum can be conducted on this system also.
引文
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