【作者】 王哲；

【导师】 章云； 贾俊香；

【作者基本信息】 沈阳农业大学 ， 农业硕士（专业学位）， 2024， 硕士

【摘要】 芥蓝（Brassica oleracea var.alboglabra）是十字花科芸薹属甘蓝种蔬菜,以花薹和嫩叶为食用器官,异花授粉作物,可通过5～6代的自交纯化的方法获得纯系,而利用游离小孢子培养技术（Isolated Microspore Culture,IMC）将获得基因型纯合的双单倍体（Doubled Haploid,DH）的时间缩短为一年。本试验旨在优化芥蓝小孢子培养技术,建立高效的小孢子培养体系,快速创制芥蓝纯合育种资源,为培育杂交新品种奠定基础。本试验以36个不同基因型的芥蓝为材料,通过添加不同浓度的激动素（Kinetin,KT）、亚甲基蓝（Methylene blue,MB）和共培养处理促进游离小孢子胚胎发生,对获得的芥蓝单倍体材料进行加倍处理,提高双单倍体的比率;对双单倍体植株自交繁殖、鉴定园艺性状,筛选优良的DH品系,并创制杂交组合。以下是主要研究结果:1.选取36个基因型芥蓝进行游离小孢子培养,其中10个基因型获得了胚状体,最高的出胚率为16.37胚·蕾^-1;为了优化芥蓝游离小孢子培养体系,在NLN培养基中添加0.2 mg·L^-1KT能显著提高3个供试材料小孢子出胚率;在NLN培养基中添加3 mg·L^-1MB处理能显著提高3个供试材料小孢子出胚率;将大白菜（CB-2）花蕾与芥蓝花蕾混合,进行小孢子共培养,以促进芥蓝小孢子胚状体的发生,进而获得更多芥蓝的再生植株,易出胚大白菜（CB-2）的花蕾数与芥蓝的花蕾数比值为2时,3个供试材料芥蓝再生植株分别增加了121、72和23株,添加大白菜（FT-5）花蕾共培养获得的再生植株都是大白菜。2.通过形态学和DNA流式细胞仪鉴定法对芥蓝再生植株进行倍性鉴定,10种基因型芥蓝的自然加倍率在4.04%～14.48%之间。对获得的芥蓝单倍体进行加倍处理。在植株的分枝腋芽处注射浓度为200μmol·L^-1戊炔草胺,间隔24 h连续处理3次效果最好,加倍率达到33.75%。3.对处于收获期芥蓝DH系进行园艺学性状调查,发现DH系间,以及DH系与供体植株间在株高、开展度、叶长、叶宽和主薹质量上差异显著;对其进行自交亲和性分析,发现均是自交不亲和系。筛选出5个综合园艺学性状优良DH系,可用于配制杂交组合。杂交组合与对照相比性状优良,‘23AW05’叶片数和叶片宽度以上性状显著优于‘ZHLX（CK）’和其他杂交组合;‘23AW06’主薹高度、主薹粗和主薹质量以上性状显著优于‘ZHLX（CK）’和其他杂交组合,杂种优势明显。更多还原

【Abstract】 Chinese kale（Brassica oleracea var.alboglabra）is a vagetable with tender leaves and stalks as its edible parts,belongs to cabbage（Brassica oleracea L.）,Brassica,Cruciferae family.It is a cross-pollinated vegetable that homozygous lines can be obtained by 5～6 times of self-pollinated.Double haploid（DH）utilizing isolated microspore culture（IMC）can be considerably decreased to one year.The aim of this experiment is to optimize the microspore culture technology of Chinese kale,establish an efficient microspore culture protocol,rapidly create homozygous breeding resources for Chinese kale,and lay the foundation for develop new hybrid varieties.This study used 36 different genotypes of Chinese kale as materials,and promoted isolated microspore culture embryogenesis by adding different concentrations of kinetin（KT）,methylene blue（MB）,and co-culture treatment,doubled the haploid to increase the ratio of doubled haploid.The horticultural features,self-compatibility index of the DH lines were done,and five DH lines was sellected for generate hybrid combinations.The primary research findings are as follows:1.36 genotypes of Chinese kale were selected for isolated microspore culture,of which10 genotypes obtained embryos.The highest rate of microspore embryogenesis was 16.37embryos per bud.In order to optimize the isolated microspore culture protocol of Chinese kale,adding 0.2 mg·L^-1KT to NLN medium can significantly improve the microspore embryogenesis rate in three test materials.Adding 3 mg·L^-1MB to NLN medium can significantly improve the microspore embryogenesis rate.Buds of Chinese cabbage（CB-2）were mixed with Chinese kale buds to promote the occurrence microspore embryogenesis in Chinese kale.When the ratio of number of buds from easily embryogenic Chinese cabbage（CB-2）to the number of flower buds in Chinese kale was set to 2,the regenerated plants of three test materials,Chinese kale,increased by 121,72,and 23 plants,respectively.The regenerated plants obtained by co-culture the flower buds of Chinese cabbage（FT-5）were all Chinese cabbage.2.Ploidy identification was carried out using morphological and DNA flow cytometry techniques.The 10 genotypes of Chinses kale had spontaneous doubling rates ranging from4.04%to 14.48%.At the axillary buds of plant branches,the injection concentration was 200μmol·L^-1.Applying propyzamide three times at 24-hour intervals produced the best results,with a doubling rate of 33.75%.3.The horticultural characteristics among DH lines and donor plants at the harvest stage were examined,it was discovered that the main stalk weight,plant height,spread length,leaf length,and leaf breadth varied significantly.The DH lines were all self-incompatibility.Five DH lines with excellent comprehensive horticultural traits were selected,which can be used to develop hybrid combinations.Compared with the control,the hybrids showed excellent traits,with‘23AW05’showing significantly better leaf number and width than‘ZHLX（CK）’and other hybrid combinations.The traits main stalk thick,main stalk weight,and main stalk quality of‘23AW06’were significantly better than those of‘ZHLX（CK）’and other hybrid combinations,having significant hybrid advantage.更多还原