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园艺学报 ›› 2011, Vol. 38 ›› Issue (11): 2099-2110.

• 蔬菜 • 上一篇    下一篇

大白菜、青花菜和叶用芥菜体细胞杂交种形态学与细胞学特性鉴定

廉玉姬*,林光哲,赵小梅   

  1. (临沂大学生命科学学院,山东临沂 276005)
  • 收稿日期:2011-08-31 修回日期:2011-10-12 出版日期:2011-11-25 发布日期:2011-11-25
  • 通讯作者: 廉玉姬

Morphological and Cytological Characterization of Somatic Hybrids Obtained by Tri-parental Protoplast Fusion in Brassica Species

LIAN Yu-ji*,LIN Guang-zhe,and ZHAO Xiao-mei   

  1. (College of Life Science,Linyi University,Linyi,Shandong 276005,China)
  • Received:2011-08-31 Revised:2011-10-12 Online:2011-11-25 Published:2011-11-25
  • Contact: LIAN Yu-ji

摘要: 为拓宽十字花科蔬菜育种种质资源,以大白菜(Brassica campestris L. ssp. pekinensis)、青花菜(B. oleracea L. var. varitalica)和叶用芥菜(B. juncea L. Czern)的子叶、下胚轴为材料,分离、纯化原生质体,采用40% 聚乙二醇(Polyethylene glycol,PEG)融合法进行原生质体融合。融合细胞在附加0.2 mg · L-1 2,4-D + 0.5 mg · L-1 6-BA + 0.1 mg · L-1 NAA + 0.1 mg · L-1 激动素(kinetin)的改良K8p培养基中液体培养,当细胞分裂至8 ~ 10细胞期时,将分裂的细胞包埋于0.15%琼脂糖,然后在添加0.3 mol · L-1蔗糖和2 mg · L-1 6-BA + 2 mg · L-1 玉米素(zeatin)+ 1 mg · L-1 NAA + 0.5 mg · L-1 kinetin的Kao培养基中诱导愈伤组织。将培养获得的936个愈伤组织转到3种不定芽诱导培养基MS + 5 mg ? L-1 zeatin + 2 mg · L-1 IAA;MS + 2 mg · L-1 zeatin + 2 mg · L-1 IAA;MS + 0.5 mg · L-1 6-BA + 0.5 mg · L-1 NAA上诱导芽分化,当芽长3 cm左右时,转到1/2 MS + 0.2 mg · L-1 NAA的生根培养基上诱导生根,共获得了96株再生植株,对其进行形态学、细胞学和分子生物学鉴定。形态学观察显示再生植株表型变化大,包括3个亲本的中间型或两个亲本的中间型;PCR技术鉴定结果显示96株杂种植株中93株为细胞质雄性不育特性;染色体计数显示,再生的植株染色体数变化范围广(2n = 38 ~ 64),但未发现染色体数总和与3种融合亲本染色体数总数(2n = 74)相一致的个体。基因组原位杂交(Genomic in situ hybridization,GISH)和扩增片段长度多态性(Amplified fragment length polymorphism,AFLP)分析结果表明,再生植株基因组中分别检测出大白菜、青花菜、叶用芥菜的DNA片段,证实了大白菜、青花菜、叶用芥菜种间体细胞杂交种的真实性。

关键词: 芸薹属, 体细胞杂交, 原生质体, 植株再生, 形态学, 基因组原位杂交(GISH)

Abstract: To obtain various genetic resources,interspecific tri-parental somatic hybridization among Brassica campestris L. ssp. pekinensis(2n = 20,AA),B. oleracea L. var. varitalica(2n = 18,CC),and B. juncea L. Czern(2n = 36,AABB)was performed. Protoplasts were isolated from hypocotyls and cotyledons,and fused using the 40% polyethylene glycol(PEG). Fused protoplast were cultured in modified K8p medium supplemented with 0.2 mg · L-1 2,4-D,0.5mg · L-1 6-BA,0.1 mg · L-1 NAA,and 0.1 mg · L-1 kinetin,and 0.4 mol · L-1 mannitol as osmoticum. At the 8–10-cell stage,divided cells were transferred to Kao’s basal medium supplemented with 0.3 mol · L-1 sucrose as carbon source,and 0.15% agarose,2 mg · L-1 6-BA,2 mg · L-1zeatin,1 mg · L-1 NAA,0.5 mg · L-1 kinetin for callus proliferation. After 35 days,when small calli reached 2–3 mm in diameter,calli were transferred to regeneration medium(MS + 5 mg · L-1 zeatin + 2 mg · L-1 IAA;MS + 2 mg · L-1 zeatin + 2 mg · L-1 IAA;MS + 0.5 mg · L-1 6-BA + 0.5 mg · L-1NAA). A total 96 regenerated plants were obtained from 932 calli. Several phenotypes were obtained in the somatic hybrids,showing differences in leaf morphology,flower size and color. Most of the plants showed intermediate characteristics between two or more of the parental species. Cytoplasmic male sterility(CMS)identified by polymerase chain reaction(PCR)amplification using Ogura CMS specific primers. Putative somatic hybrids were identified by cytological and morphological analyses. The chromosome numbers of the somatic hybrids varied from 38 to 64,which was less than the sum of the parental lines. The somatic hybiridity was confirmed by Genomic in situ hybridization(GISH)and amplified fragment length polymorphism(AFLP)analysis.

Key words: Brassica, interspecific, tri-parental somatic hybridization, protoplast, plant regeneration, morphology, genomic in situ hybridization

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