【作者】 朱彦涛；

【导师】 杨淑慎； 李殿荣；

【作者基本信息】 西北农林科技大学 ， 植物学， 2005， 硕士

【摘要】 本研究以近年来陕西省杂交油菜研究中心普通温室和大田两个栽培环境下的甘蓝型油菜品种(系)和杂交组合为材料,以Coventry J等(1988)和Teresa C T等(1999)的小孢子培养方法为基本方法,进行了较系统的油菜游离小孢子培养和DH系繁殖实验研究,其主要结果如下: 1.建立了适合于本地普通温室和大田条件的油菜小孢子培养和DH系繁殖的技术体系,使普通温室和大田材料产胚的基因型频率达到50%左右,使普通温室和大田油菜的小孢子培养体系达到实用化和规模化。通过甘蓝型油菜小孢子培养获得了200多份有价值的DH系,可供油菜育种进一步利用。2.首次对大田材料的低温预处理方式、处理时间、花序长度及其基因型对油菜小孢子胚胎发生的影响进行了较系统的评价。结果表明,30~40 cm长的花序冷藏处理10 d,小孢子仍有一定的胚胎发生能力;花序长度在0~40 cm时,用于处理的花序越长,则能进行小孢子胚胎发生的花序的有效处理时间就越长;但随着花序处理时间的延长,小孢子的产胚率有下降趋势。本结果对大田材料扩大取材规模,延长取样培养时间,有效缓解大田材料因气温高、花期短且集中与取蕾培养之间的矛盾,使尽可能多的材料培养成苗,以及对一些重要材料进行异地取材培养提供了依据。3.关于抗菌素在小孢子培养中的作用还未见报道,本文对抗菌素在油菜小孢子培养中的使用效果作出了初步评价。通过对青霉素钠、头孢噻肟钠和硫酸链霉素三种抗菌素的实验发现,抗菌素不但可以防止培养材料的污染,而且影响胚状体的产量。一般随着抗菌素浓度的增大,小孢子的产胚量逐渐降低,甚至没有胚状体的形成,而畸形胚的数量却有增加的趋势。4.提出了试管苗继代保存的方法,筛选了试管苗生根的培养基,建立了试管苗大批量移栽的关键技术。由本实验可知,通过减少继代转管次数、冷藏贮存和加入生长延缓剂MET来保存油菜单倍体试管苗的方法既简便可行,又保证了试管苗顺利越夏。培养基B5+IBA 0.5 mg/l +Suc 2%+Agar 0.9%是适合油菜试管苗生根的最佳培养基,试管苗在其中培养半月时即可用于练苗移栽。建立了一套确保油菜大批量试管苗移栽高成活率的技术体系,有效地协调了试管苗移栽过程中温度、湿度与光照等环境条件,使试管苗的移栽成活率达95%以上,为试管苗的进一步利用提供了可靠保证。5.提出了利用秋水仙碱的有效加倍方法。油菜小孢子加倍采用在NLN13培养基中加入50mg/l秋水仙碱的方法,平均加倍率为81.56%,提高了加倍效率,节约了药品和经费。幼苗和植株加倍则以浸根加倍效果较好。提出了在加倍试验中,努力提高植株加倍更多还原

【Abstract】 The system of isolated microspore culture and DH-line productivity were systematicly studied with cultivars(lines) and crossing combinations in Brassica napus L from the ordinary greenhouses and the field environment of Hybrid Rapeseed Research Center of Shaanxi Province in nearly years, with microspore culture ways of Coventry J et al(1988)and Teresa C T et al(1999)as basic ways. The main results are as following: 1. The technical system of isolated microspore culture and DH-line productivity were established in Brassica napus L to suit the conditions of ordinary greenhouses and field environment in the area. This system can make the genotype rate of microspore embryogenesis of ordinary greenhouses and field materials attain 50% or so, and make the microspore culture of ordinary greenhouses and field materials reach practicalization. Plants more than 200 of valuable DH-lines were acquired throught microspore culture in Brassica napus L . These plants are further being used in the genetic research and rapeseed breeding in Brassica napus L. 2. Affection of ways and time of low temperature pretreatment, lengths of inflorescences and genotypes of materials on embryogenesis of microspores were systemicly studied by isolated microspore culture firstly in Brassica napus L from the field environment. The result shows that microspores still had certain embryogenesis capability when the inflorescences with 30~40 cm lengths were in the cold pretreatment for 10 d. In 0~40 cm lengths of inflorescences, inflorescences pretreated were longer at length, and the efficient pretreatment time of in florescences in which microspores can conduct embryogenesis was longer. However,the embryoid yield trended to decline with the prolongation of pretreatment time of inflorescences. This result provides the basis for broadening the scale of material-collected, prolonging the time of sampling and culturing to materials from the field environment, efficient respiting the contradiction between the short and aggregate bud season of donor materials in open field as high temperature and bud-collected for culturing, making more materials cultured obtain plantlets, and getting buds and culturing their microspores for some important materials from far other places. 3. The effect that antibiotics are used in rapeseed microspore culture is given preliminary evaluation. The result shows that antibiotics can not only prevent contamination by microbes from culturing materials but also affect the embryoid yield through three kinds of antibiotics including Penicillin G sodium, Cefotaxime sodium and Streptomycin sulfate. Generally, the embryoid yield is gradually descent,even no embryogenesis capability with increase of antibiotic concentration, but the number of deformity embryoids are increasing. 4. The ways of subculture and preservation of tube-shoots are offered, the rooting medium of tube-shoots is selected and the key technique of transplant of plenty of tube-shoots is provided in Brassica napus. The result shows that the ways to perserve rapeseed haploid tube-shoots are easy and practicable throught decreasing the frequency of subculture, storing in cold condition and adding the growth retardant MET, the ways can ensure tube-shoots to live through the summer successfully. The better rooting medium of tube-shoots is B5+IBA 0.5 mg/l +Suc 2%+Agar 0.9%, tube-shoots can be transplanted when they are cultured in this medium in half month. A system to ensure high survival rates of transplant of plenty of tube-shoots is set up, the program efficient concert the conditions including temperature,humidity and light in transplant of tube-shoots, and make survival rate of transplant of tube-shoots over 95%, the program offers reliable guarantee to the further utilization of tube-shoots. 5. The efficient doubling ways of colchicine were offered. Rapeseed microspore doubling uses the ways adding 50mg/l colchicine to NLN13 medium. The average doubling rate was 81.56%. This way raises the doubling rate of colchicine and saves the chemical reagents and funds. Moreover, the effect of chromosome doubling of roots-soaked in solutions is better for shoots and plants doubling. In the experiment of colchicine doubling, the affection on doubling treatment to the survival rates and agronomic traits of plants needs to be considered when we try to improve the doubling rate of plants. In addition, the floral organ indexes of the doubled and no doubled branches of the same plants were firstly compared and statisticed. These floral organ indexes can be regarded as the morphological quantification indexes of the multiple discrimination of microspore-derived plants,and provided reference basis for identifying multiple of microspore-derived plants in Brassica napus L in early stage throught the morphological indexes.更多还原