【作者】 陈超；

【导师】 侯喜林；

【作者基本信息】 南京农业大学 ， 观赏园艺， 2011， 博士

【摘要】 本文以红掌(Anthurium andraeanum)叶片和气生根根段为外植体,分别诱导产生愈伤组织和再生团块,并对其分化过程进行了细胞学和组织化学比较研究,旨在了解愈伤组织和再生团块分化过程中细胞学变化异同及糖类和蛋白质在愈伤组织衰老与再生团块发生发育过程中的变化。活性氧与愈伤组织和再生团块的分化关系尚不明确,本文探讨了愈伤组织和团块发育过程中细胞中超氧化物含量、超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性、总谷胱甘肽过氧化物酶(GPX)活性和丙二醛含量变化,明确了其在愈伤组织和再生团块分化过程中与细胞衰老的关系。气生根来源的再生团块对秋水仙素敏感,本文以其材料,进行了秋水仙素诱导红掌四倍体的研究,这将为红掌四倍体和三倍体育种奠定基础。1.红掌气生根根段再生快繁体系的建立以红掌气生根根段为材料,诱导再生团块的产生,建立快繁系统。培养基1/2MS+6-BA 1.0mg·L-1+2，4-D 0.6 mg·L-1适于气生根的保持和繁殖,1/2 MS+6-BA 1.0mg·L-1+2.4-D0.2 mg·L-1适于诱导气生根再生团块产生,MS+6-BA 1.0 mg·L-1+2.4-D0.2 mg·L-1可使再生团块分化成苗。不论是愈伤组织,还是再生团块,出现绿色组织是分化所必需的。2,4-D、6-BA浓度及其适当比例、无机盐浓度在再生团块的诱导与分化成苗中起着重要作用。2.叶片来源愈伤组织和气生根来源再生团块细胞学研究叶片诱导所产生黄绿色、黄色和褐色3种愈伤组织的细胞核质比依次减小,细胞排列越来越疏松,细胞质变稀薄,细胞形态逐渐失去完整性,其中黄色和褐色愈伤组织细胞不断衰老最终死亡。气生根形态发生以间接器官发生为主,再生团块由气生根根段切口皮层细胞分裂和脱分化而来。再生团块内部有密集且分裂旺盛的小细胞团,其细胞为分化的原始组织,是分化产生不定芽的基础。小细胞团芽的分化发生方式属内起源,随着小细胞团的不断生长,外层细胞逐渐凋亡,最终使分化的芽突出再生团块再生成苗。再生团块的形态发生过程中,也偶见有少量胚状体产生。3.愈伤组织和再生团块发育过程中糖和蛋白质的组织化学比较研究糖的含量及其梯度变化是愈伤组织和再生团块分化的重要内在条件,蛋白质含量以及核形态是否完整是愈伤组织和再生团块分化的关键。以来源于叶片的4类愈伤组织(黄绿愈伤、深绿愈伤、金黄色愈伤和褐色愈伤)和来源于气生根的再生团块为材料,研究糖和蛋白质在愈伤组织衰老与再生团块发生发育过程中的变化。经石蜡切片PAS和汞溴酚兰组织化学染色观察发现，糖主要以颗粒状存在于愈伤组织细胞中,并有明显的围核现象,蛋白质主要集中于细胞核及其周围。在黄绿愈伤组织中,糖和蛋白质的含量从外向内显著减少；而深绿愈伤组织中梯度变化不明显。在金黄色愈伤组织中糖含量低且内外细胞间梯度小；在褐色愈伤组织中糖散乱分布。在金黄色愈伤组织中,蛋白质含量由外向内有不明显的梯度变化；在褐色愈伤组织中蛋白质则没有梯度变化。新生再生团块细胞中,糖和蛋白质含量高且由外向内梯度变化显著,再生团块膨大过程中,糖和蛋白质含量下降且内外层细胞间差异不显著。再生团块分化过程中,糖主要集中在分裂旺盛的分生细胞团和维管组织周围细胞中,蛋白质则集中于分生细胞团中。在各类愈伤组织和再生团块中均出现含有类似酚类物质的特化细胞。4.愈伤组织和团块再生体系衰老指标比较研究以愈伤组织再生体系的4种愈伤组织和气生根再生体系的再生团块为材料,比较了它们之间的超氧化物含量、保护酶活性及丙二醛水平的差异。结果表明：超氧化物含量、超氧化物歧化酶(SOD)活性及过氧化氢酶(CAT)活性均为黄绿愈伤组织最高,金黄愈伤组织和深绿愈伤组织居中、再生团块和褐色愈伤组织较低；总谷胱甘肽过氧化物酶(GPX)活性以褐色愈伤组织最高,深绿、金黄和黄绿愈伤组织次之,再生团块最低；丙二醛(MDA)含量以金黄愈伤组织最高,褐色、黄绿和深绿愈伤组织次之,再生团块最低。再生团块分化能力最强,而其细胞中超氧化物含量、MDA含量均比4种愈伤组织低,这说明细胞中自由基含量和细胞膜脂过氧化与细胞分化有一定相关性。5.秋水仙素离体诱导红掌‘亚利桑那’四倍体红掌“亚利桑那”再生团块经不同浓度的秋水仙素(0.1，0.2,0.3%)分别处理3，5和7h,然后转入附加6-BA 3mg，L-1+2,4-D 0.2mg-L-1的MS培养基上。60天后,统计团块的成活率和再生苗数。0.3%和7 h处理组合显著降低了再生团块存活率,但是秋水仙素对再生团块的分化影响并不显著。所有处理的四倍体植株的诱导率在0.2%到7.6%。最佳诱导率为用0.3%,5h处理组合。四倍体叶片气孔比二倍体的大,但密度却低于二倍体。四倍体植株叶柄粗壮,叶厚且颜色深绿。同二倍体植株相比,佛焰苞更厚更大。四倍体群体中出现双佛焰苞或花梗矮化变异。同二倍体一样,四倍体也可通过气生根产生再生团块,此技术可用于四倍体快速繁殖。更多还原

【Abstract】 Anthuriums are known as an exotic ornamental crop, they consists of about 1000 species, the Anthurium andraeanum is one of the major cultivated species and is grown for cut-flower and potted plant production. In it’s Rapid Propagation of Anthurium andraeanum via callus culture has been established and used commercially, but this method has shortcoming in loss of ability to differentiation of callus after several subcultures and formation of off-types. In order to resolve the aforementioned problems, we investigated the possibility of producing regenrable mass via aerial root in it’s and differentiation of masses. In addition, micro structural difference between different differentiation potential callus and masses were observed. and senescent index changes were also studied. The mass was sensitive to colchicines. therefore colchicines induced tetraploid were also tried in this paper.1. Establishment of Rapid Propagation System of Anthurium andraeanum via aerial root in vitroThe induction of regenrable masses from aerial root were investigated, it was found that 2,4-D had very important effect to induce regenrable masses, and 2,4-D should cooperate with 6-BA. The green tissue was essential to differentiation. The mineral salt concentration of MS played an important role in regenrabole masses induction and differentiation. The 1/2MS medium supplement with 1.0 mg·L-1 6-BA and 0.6 mg·L-12,4-D was suitable to aerial root growth, the optimal induction medium was 1/2MS+6-BA1.0 mg·L-1+2,4-D0.2 mg·L-1 and the best medium for mass differentiation was MS+6-BA1.0 mg·L-1+2,4-D0.2 mg·L-1. The masses did not showed degradation phenomenon after continued reproduction.2. Cytological Study on Callus of leaf and Regenrable Mass of Aerial Root in Anthurium andraeanumIn vitro morphogenesis of three leaf callus and regenrable masses from aerial root in Anthurium andraeanum’Arizona’were compared. It was showed that in the Kelly, yellow and brown callus, the karyoplasm ratio decrease in turn, cell arrangement became looser and looser, the shape of cell did not kept integrity. Some similar apoptotic bodies were appeared in some cells of yellow and brown callus. The morphogenesis of regenrable masses were indirect organogenesis. regenrable masses occurred from the cortex of aerial root ends. There were a lot of small and actively divided cell groups with dense nucleus in regenrable masses and meristic cells were originated from these cell groups indirect. With the proliferation of meristic cells and formation of bud primordium, the outer layer cells apoptosis gradually. Which led to bud emergence on the surface of masses. Organogenesis was the main morphogenesis pathway of masse, although a few embryoid morphogenesis was observed.3. Histochemistry Studies on Callus of Leaf and Regenrable Mass of Aerial Root in Anthurium andraeanumFour kinds of callus from leaf and Regenrable mass of aerial root in Anthurium andraeanum’Arizona’were studied by paraffin section and histochemical coloration. The results showed that the granular saccharide was mainly surrounding the nuclei. In the callus and regenrable mass protein was mainly in or around the nuclei. In kelly callus the levels of saccharide and protein decreased clearly from the outer layer of cells to the inner layer of cells. and in dark green callus the grads was unconspicuous. The contents of saccharidein and the even distribution was observed in the golden callus. The distribution of saccharide was dispersedly in the brown callus. The changes of protein were inconspicuous from the outer layer of cells to the inner layer of cells in the gold callus. However, grads of protein was little in the brown callus. There was also a visible grads of saccharide and protein when regenrable mass occurred, in the expending period of regenrable mass, the saccharide and protein were distribute evenly. In the differentiation stage of regenrable mass, the saccharide was concentrated in the cells which surrounded the dense small cell groups and the primary vascular tissue. The protein mainly concentrated in the nuclei of the cell groups. There were special cells in all types of callus and regenrable mass. The content and the grads of the saccharide and protein and the integration of the nuclei played important roles in the differentiation of callus and regenrable mass.4. Comparative Studies on Senescent Index in Two Kinds of Regenerative System in Anthurium andraeaumThe superoxide and relative enzymes between different types of callus originated from leaves and regenerable mass from aerial roots were compared. It was showed that the orders of the levels of superoxide, the activities of superoxide dismutase (SOD) and catalase (CAT) from high to low were yellow green callus> golden callus>dark green callus> regenrable mass>brown callus. The highest activities of GPX (glutathione peroxidase) were appeared in brown callus, followed by dark green, yellow green callus, golden callus and regenrable mass For malondialdehyde (MDA), the highest contents were observed in golden callus, followed by brown callus, yellow green callus, dark green callus and RMB. It was demonstrated that the activities of SOD and CAT had synergistic effects in eliminating oxidative stress, and the higher activities of SOD and CAT in mass suggested their greater ability of eliminating oxidative stress. The enzyme activities of GPX in RMB were significantly lower than that in all callus, in other hand the activities of GPX were relatively lower in yellow green callus, the lower contents of super-oxides and MDA might be the results of higher antioxidant activities. In short, rapid propagation by regenrable mass had advantages over that by callus because of their higher anti-oxidant activities.5. Induction of Anthurium andraeanum’Arizona’tetraploid by Colchicine in VitroTetraploids plants of Anthurium andraeanum’Arizona’were successfully induced after treating diploid tissue masses with colchicine. Masses originating from diploid aerial roots were treated with colchicine at three different concentrations (i.e.,0.1.0.2,0.3%) for about 3,5 and 7h, and then were transferred into Murashige & Skoog medium containing 3 mg·L-1 6-BA+0.2 mg·L-1 2,4-D. After 60 days, the survival rate and numbers of regenerative shoots were scored. The high concentration and longer duration sharply reduced survival rate. In contrast. the regeneration of plantlets was not noticeably affected by colchicine. Tetraploid plants were obtained in all treatments, but the percentage of induced tetraploids ranged from 0.2% to 7.6%. The best induction was obtained with a 5-h. treatment with 0.3% colchicine. The stomatal size of tetraploid plants was larger than in diploid plants; however, the stomatal density was lower than in diploid plants. Tetraploid plants possessed stronger petioles, thicker and deeper green leaves, and thicker and longer lived spathes in comparison with diploid plants. Abnormal spathes, such as double spathes or those lacking pedicels, were observed in tetraploid plants. Tetraploid plantlets could be regenerated via aerial roots; this technique could be applied to tetraploid plant propagation.更多还原