【作者】 王丹；

【导师】 林永波；

【作者基本信息】 东北林业大学 ， 环境科学， 2012， 硕士

【摘要】 通过花药离体培养技术进行植物单倍体育种,是一种行之有效的育种途径。据报道,用花药培养单倍体植物的试验已在曼陀罗、烟草、水稻、甘蓝、鸭跖草等植物上获得成功。根据现有资料,花药产生单倍体植物可通过两种方式,一种是花粉经过类似胚胎发育的过程,形成花粉胚,由胚直接形成单倍体植物,如烟草、曼陀罗；另一种是花粉先形成愈伤组织,再从愈伤组织诱导单倍体植株,如水稻、甘蓝。本文通过对红王子锦带花药的两种离体发育方式进行细胞学观测与统计分析,筛选出适宜红王子锦带花粉启动分裂的因子、适宜的花粉发育时期、适宜的接种时期、适宜的培养基组合等条件。从而为缩短育种期限、染色体分析及单倍体在遗传和育种上的利用等研究产生一定作用。主要研究内容和结果如下：1花药愈伤组织诱导培养基的筛选。试验结果表明,红王子锦带花药在1/3MS+6-BA (0.5mg/L)+NAA (2.0mg/L)+SUC (2%)的培养基上,温度28℃,pH=5.8,暗培养,每天24h的条件下,愈伤组织诱导效果最好,平均诱导率为57.45%。2花药胚状体诱导培养基的筛选。试验结果表明,红王子锦带花药在1/3MS+6-BA(2.0mg/L)+NAA(1.0mg/L)+SUC(1%)的培养基上,温度28℃,pH=5.8,暗培养,每天24h的条件下,胚状体诱导效果最好,平均诱导率为15.15%。3适宜花粉启动分裂的发育时期。适宜花粉启动分裂发育成愈伤组织或胚状体的时期均为四分体时期。在四分体时期出愈率和出胚率分别为53.2%和6.73%,均大大高于单核期和单核靠边期。4适宜诱导的接种时期。适宜花粉启动分裂发育成愈伤组织或胚状体的接种时期均为始花期6月及盛花期7月,在末花期8月不适宜接种培养。5低温预处理对花药诱导率的影响。将花药放入4℃C的冰箱内,进行不同天数的低温预处理,结果显示,预处理天数最长(6天)的花药愈伤组织及胚状体的诱导率最高,为41.98%,未经低温预处理的花药诱导率最低,仅为12.47%。6适宜花粉启动分裂的主要因子。适宜花粉分裂成愈伤组织的主要因子为MS基本培养基与蔗糖,MS基本培养基和蔗糖的浓度愈低,出愈率愈高；适宜花粉分裂成胚状体的主要因子为MS基本培养基与6-BA,MS基本培养基的浓度愈低,6-BA的浓度愈高,出胚率愈高。更多还原

【Abstract】 The technology of anther culture in vitro was an effective way for plant haploid breeding.lt was reported that the test of anther culture haploid plant had been successfully on Datura stramonium,Nicotiana tabacum L., Oryza sativa, Brassica oleracea var. acphala f. tricolor, Commelina communis L. and so on.According to the existing data,anther produced haploid plant by two means,one was pollen by similar embryos development process,formed pollen embryo,then haploid plant, such as Nicotiana tabacum, Datura stramonium;The other way was that pollen first formed callus,then induced haploid plant,such as Oryza sativa, Brassica oleracea var. acphala f. tricolor.The article through two Weigela florida cv.’Red Prince’development system in vitro to cytologic observation and statistical analysis,screened out factors of pollen starting division,suitable pollen development data,suitable inoculating data,suitable medium combination and other conditions.Thus for the effect on shortening breeding term,chromosome analysis and haploid using on genetic and breeding.The main contents and results of this study are as follows:1The selection of anther callus on inducing medium.The results showed that the medium1/3MS+6-BA(0.5mg/L)+NAA(2.0mg/L)+SUC(2%) is best,which is favorable to induce the anther callus on Weigela florida cv.’Red Prince’with temperature28℃,pH5.8,dark culture and24h per day.The average induction rate in callus is57.45percent.2The selection of pollen embryos on inducing medium.The results showed that the medium1/3MS+6-BA(2.0mg/L)+NAA(1.0mg/L)+SUC(1%) is best,which is favorable to induce the pollen embryos on Weigela florida cv.’Red Prince’with temperature28℃,pH5.8,dark culture and24h per day.The average induction rate in callus is15.15percent.3The suitable development data of pollen starting division.The suitable data of pollen starting division which developed callus or embryos was tetrad stage.The rate of callus and embryos was respectively53.2percent and6.73percent, all greatly higher than the uninucleate stage and the late uninucleate stage.4The suitable inoculating data of induction. The suitable inoculating data of pollen starting division which developed callus or embryos were all in six month at the beginning of flowering period and in seven month at the full flowering period,it was not suitable for eight month at the end of flowering period to inoculating culture. 5The influence of anther induction on lower temperature pretreatment.Put the anthers into the refrigerator of4℃,lower temperature pretreatment was processed in different days.The results showed that the highest induction rate of anther callus and embryos was the longest pretreatment(six days),which was41.98percent.The anther induction rate was only12.47percent without lower temperature pretreatment.6The main factors of suitable pollen starting division.The suitable factors for callus of pollen starting division were MS basic medium and sucrose.The lower the level of MS basic medium and sucrose,the higher the rate of callus; The suitable factors for embryos of pollen starting division were MS basic medium and6-BA.The lower the level of MS basic medium,the higher rate of embryos.the higher the level of6-BA,the higher the rate of embryos.更多还原