苜蓿花药组织培养及DH群体构建

Haploid could be obtained by alfalfa anther culture. Some characters of haploid were controlled by recessive genes, which could well express after chromosome doubling, and then the new homozygous inbred lines（DH） would be obtained in a relatively short time. DH groups were valuable materials in choosing the strong superiority crosses of high yield and quality, and it played an important role in acceleting the purification of germplasm materials and improving the ablilty of breeding innovation, which has been widely used in crop breeding. The main problem in the utilization of alfalfa male sterile heterosis were lacking of gene homozygous in parent materials and hybrid. Given all this, the male sterile and its corresponding fertile plants of alfalfa were considered as the experimental materials in the study. The anther culture technique will be used to cultivate alfalfa regenerated plants. The haploid are going to detect by cytogenetic and flow-cytometric analysis from those regenerated plants. The homozygous amphidiploid plants can be obtained after colchicine treatments. the DNA methylation and SRAP molecular marker technique and FCM would be use to detect the homozygote and heterozygote rapidly and accurately.At last, the DH group of alfalfa will be established. All of the work will lay a foundation for cultivating excellent restorer lines and hybrid seed production of alfalfa male sterile.

利用苜蓿花药组织培养技术获得单倍体，若其中由隐性基因控制的性状经染色体加倍后仍能很好表现，可在较短时间内获得新的纯合系（DH）。DH系是选配强优势组合的宝贵材料，在加速种质材料纯化及提升育种创新能力方面具有重要的利用价值，目前该技术已在农作物育种中广泛应用。本项目针对苜蓿雄性不育杂种优势育种中缺少基因型纯合的父本材料、杂优组合少等问题，以苜蓿雄性不育株及其对应可育株为材料，通过花药组织培养技术获得再生植株，利用细胞遗传学和流式细胞术鉴定方法筛选单倍体。经秋水仙素加倍植株，通过DNA甲基化结合SRAP分子标记技术和流式细胞术检测倍性，获得双二倍体植株，并构建苜蓿DH群体。为培育优良恢复系及苜蓿雄性不育杂交制种奠定基础。

通过苜蓿花药或花粉培养获得单倍体，进一步利用纯系材料杂交育种，可充分发挥杂种优势，缩短育种年限并提高育种效率。本研究以花药为材料对草原1号杂花苜蓿、呼伦贝尔黄花苜蓿和新疆大叶紫花苜蓿进行花粉组织培养，通过固体与液体悬浮培养基、不同激素配比进行正交试验诱导花药愈伤组织，分化和生根培养后获得再生植株，建立适宜的花药组培再生体系；再生植株先通过间接鉴定（株高，主茎粗，再生速度，SPAD值）聚类筛选，对筛选出单倍体植株进行气孔数初选、根尖染色体和流式细胞仪鉴定，确定染色体倍性。主要研究结果如下：.花药愈伤组织的诱导，草原1号杂花苜蓿固体培养基优于液体悬浮培养基，而呼伦贝尔黄花苜蓿与新疆大叶紫花苜蓿液体悬浮培养基优于固体培养基，出愈率最高值分别为83.33%、70.00%、83.33%。根据正交试验出愈率极差的分析，草原1号杂花苜蓿适宜的固体培养基为B5 + 0.4 mg/L NAA+ 0.75 mg/L 2,4-D + 1.0 mg/L KT + 0.75 mg/L 6-BA；呼伦贝尔黄花苜蓿适宜的液体悬浮培养基为B5 + 0.4 mg/L NAA + 0.50 mg/L 2,4-D + 3.0 mg/L KT +0.25 mg/L 6-BA；新疆大叶紫花苜蓿适宜的液体悬浮培养基为MS + 0.2 mg/L NAA+ 2.0 mg/L 2,4-D + 3.0 mg/LKT+ 0.5mg/L 6-BA；草原1号杂花苜蓿和新疆大叶紫花苜蓿花药愈伤分化培养的适宜培养基为MS + 1.0 mg/L KT + 0.2 mg/L NAA，分化率58.33%-73.33%；呼伦贝尔黄花苜蓿花药愈伤分化培养的适宜培养基为MS + 1.0 mg/L 2,4-D + 0.5 mg/L 6-BA，分化率高达88%。.对75株苜蓿花药组织培养再生植株，利用间接的倍性鉴定方法判断其中单倍体植株有8株，雄性可育与雄性不育的花培植株各有4株，占总株数的10.7%。结合染色体数鉴定结果，初步判定有单倍体或双单倍体植株4株。在各鉴定方法中，株高、气孔数和保卫细胞叶绿体数结合起来分析倍性较为准确。.草原1号杂花苜蓿雄性不育植株、呼伦贝尔黄花苜蓿及新疆大叶紫花苜蓿花药培育再生植株中单倍体植株分别为4株、4株、7株，分别占各自总株数的8.51%、27%、14%。