【作者】 李红民；

【导师】 马晖玲；

【作者基本信息】 甘肃农业大学 ， 草业科学， 2009， 硕士

【摘要】 匍匐翦股颖(Agrostis stolonifera L.)是一种很重要的多年生冷地型草坪草,具有耐修剪、观赏性高等优点,常用于高尔夫球场果岭及高档草坪中。本研究探究了诱导匍匐翦股颖愈伤组织的最佳条件,建立了高频再生体系。并对匍匐翦股颖愈伤组织进行了玻璃化超低温保存,为匍匐翦股颖细胞工程和基因工程奠定了理论和实践基础,同时对匍匐翦股颖属种质资源长期保存提供了依据。主要研究结果如下:1.建立了匍匐翦剪股颖再生体系:分别以A-4、A-1、帕特三个品种成熟种子为外植体进行愈伤组织诱导,其中品种A-4的愈伤组织诱导率最高,可达74.7%;2,4-D是影响愈伤组织诱导的最重要激素,以4 mg/L为宜,愈伤组织诱导率为73.2%;在MS+4mg/L2,4-D+0.3mg/L6-BA+蔗糖30g/L+琼脂7g/L中可显著提高愈伤组织诱导率,愈伤组织诱导率可达78.6%;继代培养条件:MS+2mg/L2,4-D+0.1mg/L6-BA +0.3mg/LABA+ 500 mg/L水解酪蛋白中对愈伤组织进行继代培养,胚性愈伤组织发生率可达36.8%;芽分化:在基本培养基中添加2mg/L 6-BA,芽的分化率在80%以上;根的诱导:在1/2MS培养基中添加0.5 mg/LNAA;2.探索了玻璃化超低温保存体系:将继代两次的愈伤组织在4℃低温度下预培养5天,常温下用装载液(2mol/l甘油+0.4 mol/l蔗糖)过渡10min,再于0℃下用玻璃化溶液PVS2处理50min,换成新鲜的PVS2迅速投入液氮中保存一小时以上,取出后在30℃水浴中化冻,用MS+1.0mol/l蔗糖溶液洗涤3次,每次10min,细胞存活率可达85.6%。3.超低温前后愈伤组织,过氧化物酶同工酶酶谱没有发生变化,染色体数目没有改变。该研究结果为匍匐翦股颖属植物种质资源的长期保存提供了基础数据。更多还原

【Abstract】 Agrostis stolonifera L., a significant-grown cool season turf garss, with beautiful colour and the adventage of cut-resistance,Commonly used in the golf courses of the green and High-grade lawn. This study explores the best conditions of induction callus and establish high efficiency regeneration system. The procedure for cryopreservation by vitrification was preliminarily developed in Creeping bentgrass calli.It provide theoretical and practical basis for cell project and gene project of Creeping bentgrass. At the same time, it provided the basis for Long-term preservation of Agrostis stolonifera L. germplasm resources. The results showed that:1. The regeneration system of Creeping Bentgrass have been estabilished. With A- 4、A-1and pate for the three varieties of explant on callus induction, A-4 have the highest rate of callus induction; 2,4-D is the the most important hormone affecting callus induction, It has shown that 4 mg/L 2,4-D was optimal for callus induction. It can significantly improve the induction rate, which show that MS medium appended with 4mg/L2,4-D , 0.3mg/L 6-BA, 30g/L sucrose and 7g/L agar can give highest rate of callus induction; Callus subculture: MS medium supplemented with 2,4-D2.0 mg/L, 6-BA0.1 mg/L,ABA 0.3mg/Land CH 500mg/L is the most effective subculture medium. The best differentiation medium is MS+6-BA2.0 mg/L,the inductivity can reach 80%.;Roots induction: The suggested medium is 1/2MS+0.5 mg/LNAA2. The procedure for cryopreservation by vitrification:After subcultured twice, the callus was pre-cultured five days at 4℃,and then the excised calli were loaded with 2 mol/l glycerol and 0.4 mol/l sucrose.then the calli were exposed to PVS2 for 50 min at the zero temperature. after changing the solution with fresh PVS2, the calli were put into the liquid nitrogen at least one hour. the calli were thawed rapidly in a 30℃water bath. then they were washed with the MS medium with 1.0 mol/lsucrose for 3 times and each 10 minutes. The survival of cells can reached 85.6%. 3. Before and afte cryopreservation calli,The Peroxidase isozyme zymogram and chromosomes is not changed. The result provided a feasible proof of conservation Agrostis stolonifera L..through cryopreservation of in vitro shoot-tips by vitrification for long term更多还原