【作者】 曲同宝；

【导师】 王丕武；

【作者基本信息】 吉林农业大学 ， 作物遗传育种， 2004， 硕士

【摘要】 羊草（Leymus chinensis／Aneurolepidium chinensis）是分布于我国北方天然草地面积较广、营养价值较高而且较耐盐碱的优良的禾本科牧草，是经济价值较高的草原类型。羊草具有耐寒、耐旱、耐湿、耐践踏、耐土壤瘠薄和一定的耐盐碱能力，适应范围很广。但是由于近年来人们的过度开发和利用，造成天然羊草草原的退化。为了保护羊草资源，提高草地生产力，探索改良盐碱化草地的技术和途径已成为必然趋势，如何利用大面积盐碱地是我们全人类迫切需要解决的问题。 羊草是建立人工草地和治理盐碱化草地的理想草种，虽具有一定的抗盐碱能力，但一直未能很好地发挥改良盐碱地的作用。根据羊草生育规律及生物学特性，进行栽培、利用，充分发挥羊草具有的耐盐碱能力，不断培育出高度耐盐碱的羊草新品种，才是开发利用羊草草地资源的根本对策。 采用传统的育种方法选育耐盐品种极为缓慢。随着分子生物学技术的发展，人们可以用基因工程技术提高植物的耐盐碱性。提高植物抗盐性最根本、最经济、最有效的方法是充分利用抗盐基因，培育新的高抗盐品种。 本文利用野生羊草种子采用组织培养方法，探索羊草的生长发育规律及相关的生物学特性，开展羊草组织培养和遗传转化研究，建立了高效的羊草遗传转化受体系统，为培育出品质优良又高度抗盐碱的羊草新品种打下良好基础；并在此基础上，进行了羊草愈伤组织的抗旱、抗盐碱敏感性研究，确定羊草愈伤组织在盐碱培养基上的最大忍受强度，通过在高盐碱浓度培养基上筛选，希望获得羊草耐盐突变体植株；首次通过基因枪方法将BADH基因转入羊草受体系统中，并对转化结果进行了初步检测。取得的主要研究结果如下： 一、建立并优化了羊草遗传转化受体系统 1．羊草愈伤组织的获得 羊草的成熟胚（种子）及根茎是诱导愈伤组织理想外植体。外植体的灭菌时，首先用70％乙醇浸泡5min，然后在1g／L升汞溶液中浸泡15min，这样可以达到灭菌的最佳效果。羊草种子具有休眠特性，经过低温处理后有利于种子萌发，随着低温处理天数增加，种子萌发率也升高；变温更有利于羊草种子萌发。用100ppm赤霉素（GA₃）处理休眠的羊草种子时，处理48小时为打破休眠、促进萌发的最佳时间，种子的萌发率可达16％。 为了提高成熟胚的胚性愈伤组织的诱导率，设计了不同的培养基及不同的激素浓度。羊草愈伤组织诱导采用MB（MS+B5有机）培养基，当培养基中2，4-D的浓度吉林农业大学硕士学位论文羊草遗传转化受体系统的建立及转BADH基因的研究为2 .Omg/L时诱导率为20%，本实验中，羊草愈伤组织诱导的最适2，4一D浓度为2.smg/L，诱导率达到了24%。2.羊草愈伤组织的继代培养 羊草愈伤组织继代培养的最佳2，4一D浓度为2 .omg/L .MB和Ms培养基是较适宜的羊草愈伤组织继代培养基。在继代培养中蔗糖较适宜的浓度为409/1。培养基中脯氛酸浓度为2 50omg/L，是羊草愈伤组织的生长以及胚性状态保持较适宜的浓度。3.羊草愈伤组织的分化及再生 羊草再生系统属于胚胎发生型，随继代培养时间的延长，愈伤组织的分化能力将迅速下降，继代培养6个月后分化能力基本丧失，所以用于分化的羊草愈伤组织继代时间不宜过长。 培养基中加入0 .smg/L6一BA为羊草愈伤组织分化最佳条件，分化率达到了2 6 .67%。生根培养基中加入NAA的最适宜浓度为2 .omg/L。如果在愈伤组织分化前进行预分化10一30天，可以明显提供分化出苗率。二、羊草愈伤组织耐盐敏感性测定 综合增重指数和愈伤组织生长状态两种因素考虑，羊草愈伤组织对中性盐（Nael）的可耐受最大强度为1 50二01/L，对碱性盐（NaHeo。与NaZeo。）的可耐受最大强度为4Inmol/L，对PH值的可耐受最大强度为8 .0。羊草愈伤组织在高盐浓度条件下，可以筛选出耐盐突变体植株。从4Inmol/L开始筛选，通过逐步提高碱性盐浓度的方法，最终获得了碱性盐筛选浓度为12¹/L耐盐植株的愈伤组织，并成功分化出5株幼苗。经形态学观察发现其叶色浓绿，株型低矮，茎叶直立，与一般分化植株明显不同。三、基因枪转化参数研究。 当培养基中的kana功yC in浓度达到140啊/L以上时，羊草愈伤组织增重指数明显下降，达到1 50mg几时，羊草愈伤组织已经基本停止生长，羊草愈伤组织对卡那霉素的敏感浓度为1 50mg/L。羊草遗传转化可能不适宜用农杆菌浸染法。 本实验中用于基因枪转化的杭盐碱基因为甜菜碱醛脱氢酶（BADH）基因。PCR及电泳结果表明，BADH基因已被整合到羊草染色体组中。采用基因枪转化研究，不同的轰击距离（6cln、gcm、12Cm）转化效率不同。随着轰击距离的加大，转化效率逐渐降低，当距离为6cm时，枪击后细胞受损严重，褐化较多，因此9 Cm轰击距离较为适宜。DNA用量为每枪6ul（含3。。ug金粉和1 .ZugDNA），11oopsix吉林农业大学硕士学位论文羊草遗传转化受体系统的建立及转BADH基因的研究9。。的压力x靶距组合，转化效果最好。在轰击距离为gctn的条件下，轰击1、2、3次，转化效率分别为88 .13%，41 .12%和18 .21%，轰击1次时转化效果最好。 植物转化研究中，高效遗传转化受体系统的建立是转?更多还原

【Abstract】 Leymus chinensis is an important grass of the Gramineae family. It widely grows in the natural grassland of Northern China. Its value of nutrition is very high and it can grow very well in the saline-alkaline field. There is important economical value. Leymus chinensis whose adaptability is strong can stand coldness, drought, wet, trample, poor soil conditions and some saline-alkaline stress. But the grassland has been excessively exploited, which makes the grassland degradative. In order to protect the resource of Leymus chinensis and improve the productivity of grassland, it is necessary to seek the new technologies and approaches of melioration. It is an urgent problem to solve how to utilize large area of saline-alkaline field.Leymus chinensis is an ideal grass to build meadow and control the saline-alkaline field. Although Leymus chinensis can endure saline-alkaline stress, it can’t affect melioration of the saline-alkaline field. According to law of reproductive and characteristic of biology, we can plant and approve Leymus chinensis. We should utilize the capacity of bearing saline-alkaline and breed some new kinds of species enduring saline-alkaline stress, it is basic route to control saline-alkaline.It is extremely slow to select and breed species which bear salt by traditional methods. With the development of molecular biology, we can use technology of gene engineering to improve endurance of saline-alkaline. The most efficient and ultimate method is to utilize saline-alkaline resisted gene in order to breed high-resisted species.This paper studied the law and peculiarity of Leymus chinensis and optimized the tissue culture system. The high efficient regeneration system firstly is founded, which makes it a good foundation to breed the new species which good characteristic and ability to endure saline-alkaline stress. The tissue culture system of mature embryos of Leymus chinensis was optimized. The sensitive characteristic on saline-alkaline of Leymus chinensis calli was studied; first assure Leymus chinensis calli enduring on the saline-alkaline medium. We hope to get saline-endured mutation by selecting with the high-concentration saline-alkaline medium. Studies have been done on genetic transformation and ways of saline-endurance. BADH gene was transferred into the regenerated system of Leymus chinensis by gene-gun and then examined. The results of study were acquired as follows:1. The tissue culture system of mature embryos on Leymus chinensis was optimized.(1) Acquisition of Leymus chinensis calliMature embryo (seed) and root-stock is the ideal part to induce calli. It can be effectively kill germ by using 70% alcohol for 5 minutes and then in 1g/L Hgcl2 for 15 minutes. There is the characteristic of sleeping for Leymus chinensis seed. Low temperature contributed to germination. For some days accumulation germination rate increased. Temperature varieties contributed greatly to germination. 48 hours is the best time for germination by using 100ppm GA3, and germination rate can get to 16%.In order to improve the embryogenic calli induction rate of mature embryos, different media with different hormones were used in the embryogenic calli induction. The experiment results indicated that the selection of suitable medium is the basis to improve the induction rate and the quantity of embryogenic calli inducing from mature embryos. The hormone treatments had certain effects on the calli induction which lead to the significant difference. The mature calli induction of different media is quite different. MB gain very well induction efficiency. Among the difference of hormones, when concentration of 2,4-D was 2.5mg/L it could obviously enhance efficiency of induction and embryos induction.(2) Leymus chinensis calli subcultureIn the experiment, MB and MS media could improve the growth of mature calli and could obtain embryogenesis calli at last. The 2.0mg/L is the best in Leymus chinensis calli subculture when using 2,4-D. Concentration of sugar and proline should be 40g/L and 2500mg/L in su更多还原