板栗组织培养及褐变研究

【作者】 罗丽华；

【导师】 苏冬梅；

【作者基本信息】 中南林学院 ， 植物学， 2004， 硕士

【摘要】 本实验以板栗品种韶十八为主要材料，其余十个品种作为辅助研究材料，通过组织培养技术，利用不同品种、不同外植体部位的板栗，研究板栗愈伤组织诱导、继代培养和植株再生的适宜培养基。结果表明，不同的品种适宜培养基不同，韶十八的适宜培养基为WPM+6-BA_0.5+NAA_0.2，以其胚培养诱导率较高，成愈率为79.5％。 由于板栗单宁含量很高，故在板栗组织培养过程（无论是初代培养还是继代培养）中，外植体都易发生褐变，导致培养物生理功能衰退，严重阻碍了板栗组织培养的正常进行。本文以韶十八愈伤组织为材料，对板栗外植体褐变相关的生理因子及影响因素进行了系统研究，目的在于了解减轻板栗褐变的有效措施，从理论上认识外植体组织褐变；通过研究各因素对褐变的影响，找到影响褐变的主导因子，以及减轻褐变的有效方法，以期为板栗褐变提供理论依据和技术支持。本试验通过研究不同浓度不同种类的抗褐变剂、不同浓度的AgNO₃、不同光照条件、不同温度、不同pH值对愈伤组织褐变的影响，研究了各个因素对褐变程度的影响，结果表明：在6g.L^-1PVP、10mg.L^-1AgNO₃、前期暗处理、19℃、pH值为5.8～6.0条件下培养，有利于减轻褐变。另外，对不同品种不同材料的过氧化物同工酶谱进行研究，可以看出不同品种之间的亲缘关系，以及不同材料的分化程度。 本文对褐变的机理进行初步研究，分别统计和测定培养期间褐变率、PPO活性、POD活性，研究培养过程中酶的活性变化与外植体褐变的关系，结果表明：板栗组织培养过程中，PPO活性与褐变存在显著的相关关系，PPO活性高的处理其褐变率也较高，POD活性与褐变之间无相关性。试验研究不同伤害程度与愈伤组织总酚含量之间的关系，发现褐变不仅是因为原有的酚类与PPO反应，而且由于伤害作用产生了新的酚类化合物，从而加剧了褐变。更多还原

【Abstract】 Castanea mollissima species shao shiba was the main research material in this experiment, other ten species were assistance research material. I researched the optimum medium of the different species and explant for inducing callus, subculture and regenerate on Castanea mollissima through tissue culture technique. The results showed that different species of Castanea mollissima need different medium, and WPM + 6-BA0.5+NAA0.2 was shao shiba’s optimum inducing medium, and the inducement rate of embryo (79.5%) was higher than other explants. The subculture medium of shao shiba was WPM+6-BA0.5-1.0+NAA0.2.Because of containing much tannin, the explant of Castanea mollissima brown seriously, which interfered with normal propagation of Castanea mollissima tissue culture. Take shao shiba as research material, I studied the mechanism of explant browning of Castanea mollissima and factors influencing the explant browning in subculture. Then 1 find out the efficiency measures of alleviating browning.I researched the effect on browning of different concentration added materials , different concentration of AgNO3, light condition, temperature and pH. The result suggested that the conditions of 6g. L-1 PVP, 10mg.L-1 AgNO3, prophase dark treatment, 19℃ and pH 5.8 - 6.0 avail to lighten brownimg. Electrophoretic character of POD isozyme of the different species’ different materials were studied, 1 found out the relation of different species and the differentiation degree of different materials.According to the browning phenomenon of Castanea mollissima in plant tissue culture and the activity of the peroxidase and polyphenolic oxidase, the results of my study indicated that the activity of PPO has relativity with the browning of the callus, that is the higher activity of enzyme,the more severity of browning.But POD was not as PPO.At the same time, analyzing correlation reveals that the correlation relationship of different harm degree and total phenol content is significant, 1 conclude that harm produce more phenolic, so total phenolic content deteriorate the degree of browning.更多还原