青花菜与白菜间体细胞杂种获得与遗传特性鉴定

Morphological, cytological, and molecular characterization of hybrids and their progenies derived from the somatic hybridization of Brassica campestris and Brassica oleracea

摘要:

为获得芸薹属白菜Brassica campestris与青花菜Brassica oleracea var. botrytis的种间体细胞杂交体，以青花菜和白菜的子叶与下胚轴为材料，分离制备原生质体，用40％聚乙二醇 (Polyethylene glycol，PEG) 进行原生质体融合。融合细胞在以0.3 mol/L 蔗糖、0.3 mol/L葡萄糖为渗透稳定剂，附加0.2 mg/L 2,4-D+0.5 mg/L 6-苄氨基嘌呤 (6-BA) +0.1 mg/L 1-萘乙酸 (NAA) +0.1 mg/L激动素 (Kinetin，Kin) 的改良K8p培养基中液体浅层培养。将包埋于0.1％琼脂糖的8~10个细胞期的细胞在添加0.3 mol/L蔗糖和2 mg/L 6-BA+2 mg/L玉米素 (Zeatin，ZEA) +1 mg/L NAA+0.5 mg/L Kin的Kao培养基中诱导愈伤组织。愈伤组织转到MS+5 mg/L ZEA+2 mg/L IAA诱导不定芽。将长1～2 cm的不定芽转到1/2 MS+0.2 mg/L NAA诱导生根。将生根的植株转移到花盆，并对其杂种性质进行形态学、细胞学和分子生物学鉴定。结果表明，融合细胞培养2～7 d后发生第1次分裂，培养35 d后植板率为0.66％，不定芽再生率达3.7％。形态学观察显示，绝大多数再生植株的叶面积较大，株型和叶型为两种杂交亲本的中间型。染色体计数结果显示，再生植株染色体数目为2n=38。流式细胞仪测定DNA含量显示，再生植株DNA含量是亲本之和。随机扩增多态性DNA (Random amplified polymorphic DNA，RAPD) 和基因组原位杂交 (Genomic in situ hybridization，GISH) 分析结果证明再生植株具有双亲基因组。体细胞杂种花粉育性比较低，杂交、回交后其育性逐渐获得恢复。

Abstract:

In order to produce interspecific somatic hybrids between Brassica campestris (2n?=?20, AA) and Brassica oleracea (2n=18, CC), we isolated protoplasts from cotyledons and hypocotyls of young seedlings, and fused by 40% polyethylene glycol (PEG). Fused cells were cultured in modified K8p liquid medium supplemented with 0.2 mg/L 2,4-dichorophenoxyacetic acid (2,4-D) +0.5?mg/L 6-benzylaminopurine (6-BA)+0.1 mg/L naphthaleneacetic acid (NAA)+ 0.1 mg/L Kinetin (Kin), 0.3 mol/L sucrose and 0.3 mol/L glucose were used as osmoticum. At the eight-to ten-cell stage, divided cells were transferred to Kao’s basal medium supplemented with 0.3 mol/L sucrose as carbon source and 0.1% agarose, 2 mg/L 6-BA+ 2 mg/L Zeatin (ZEA)+1 mg/L NAA+ 0.5 mg/L Kin for callus induction. After 35 days, when small calli reached 2–3 mm in diameter, calli were transferred to regeneration medium containing 5 mg/L Zeatin (ZEA) and 2mg/L indole-3-acetic acid (IAA). After the length of the shoots reached 1–2 cm, the shoots were transferred to 1/2 MS+0.2 mg/L NAA for root induction. Morphological, cytological and molecular biological analysis methods were used for identification of somatic hybrids. The results showed that, the first cell division occurred during 2–7 days of culture. Five weeks after culture initiation, the plating efficiency attained 0.66%. Finally, the shoot regeneration frequency was 3.7%. A total of eleven regenerated plants were obtained and verified as somatic hybrids by morphological observation and flow cytometry. Cytological studies showed that all tested plants had a chromosome number of 38, the sum of both parents. Hybridity was also confirmed by randomly amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analysis, indicating that these regenerated plants were all true hybrids of B. campestris and B. oleracea. All amphidiploid somatic hybrids showed low pollen fertility. Pollen fertility was gradually recovered in the first and second progenies.