通过不对称体细胞杂交向小麦转移青苗碱谷DNA(英文)

普通小麦“济南177”(Triticum aestzvum L.cv.Jinan 177)经继代培养和选择,形成了两种性质不同的愈伤组织,一种生长迅速,易于形成悬浮系,游离的原生质体具有旺盛的分裂能力,但不能分化,称为Cha 9;另一种具有一定分化能力,但游离的原生质体分裂能力低,称为176。二者之一来源的原生质体与紫外线照射的青苗碱谷原生质体融合均不能获得再生植株。而将来源于Cha 9和176的两种原生质体混合,与经紫外线照射的青苗碱谷原生质体在PEG诱导下融合,融合产物再生了大量植株。再生的愈伤组织及植株经表型、细胞学、同工酶、RAPD分析,证明了其杂种性质,用小麦叶绿体特异的简单重复序列(SSR)引物分析了再生杂种的叶绿体遗传组成情况。在不同的杂种克隆中,同时带有Cha 9和176的遗传物质并含有供体核及胞质基因组的克隆4具有较高的分化能力,再生了大量生长旺盛的完整植株。     

混合小麦亲本与新麦草不对称体细胞杂交体系的建立

以小麦品种济南177悬浮细胞系来源的原生质体与同品系胚性愈伤组织制备的原生质体混合后作为受体;以经过380μＷ/ｃｍ²紫外线照射1min、2min的新麦草原生质体分别作为供体,用PEG法诱导融合。组合Ⅰ(176+cha9+新麦草UV 1min)获得16个再生克隆。经过形态学、同工酶、染色体和RAPD分析,确定其全部为属间体细胞杂种。其中的5个克隆再生杂种植株。用7对小麦SSR引物对杂种克隆的叶绿体基因组进行了分析;组合Ⅱ(176+cha9+新麦草UV 2min)只获得3个克隆,且逐渐褐化死亡。表明以小麦济南177的两种培养细胞混合作受体的融合体系有利于杂种的获得及再生;紫外线对融合产物的生长发育有明显的剂量效应。     

小麦与燕麦不对称体细胞杂交的研究

以小麦品种济南177的悬浮细胞系(长期继代培养已丧失分化能力)来源的原生质体混合同品种胚性愈伤组织(分化能力较强, 约70%)制备的原生质体为受体, 以经300 mW/cm²紫外线照射0.5, 1, 2, 3, 5 min的普通燕麦愈伤组织(分化频率很低, 约10%)原生质体作供体, 用PEG法诱导融合. 可高频率地获得体细胞杂种细胞系, 并分化获得绿色正常的再生植株, 经荧光原位杂交、 同工酶及5S rDNA间隔序列分析, 确认了它们为体细胞杂种. 单独使用小麦胚性悬浮系或愈伤组织为受体获得的杂种克隆均未能得到绿色植株.     

普通小麦与簇毛麦原生质体的紫外线融合

从来源于普通小麦品种济南177（Triticum aestivum cv.Jinan 177）悬浮细胞系的原生质体与来源于簇毛麦（Haynaldia villosa）胚性愈伤组织的原生质体融合获得体细胞杂种。供体簇毛麦原生质体在融合之前用紫外线照射30s或1min,紫外线剂量为360Цw/cm^2。仅由紫外线照射30s的组合获得再生愈伤组织克隆。细胞学、生物化学及PCR分析结果证实了再生克隆的杂种性质。用线粒体基因特异的探针进行的RFLP分析的结果表明,杂种中含有融合双亲的线粒体并且发生了重组。由杂种愈伤组织再生得到白化苗。讨论了紫外线对融合产物的影响。     

单倍体小麦与羊草的属间体细胞杂交

以失去植株再生能力的小麦单倍体愈伤组织和羊草二倍体愈伤组织为材料,游离原生质体,并用紫外线自理２羊草原生质体,用ＰＥＧ法融合,对融合克隆进行染色体和同工酶分析,在已贩２６个克隆中有２１个是杂种,其中有一个克隆再生出短命小植株,结果 体小麦与二倍体草的不对称融合虽然再生互补效应不如二倍体小麦,然而杂种优先生长的现象仍然比较明显。如果改善实验条件和双亲原始的再生能力,这种融合方式仍然可以利用。     

普通小麦与玉米不对称体细胞杂交的研究

以长期继代培养,经分化实验证明无分化能力的普通小麦（Triticum aestivum L.)济南177原生质体为受体,以经380uW/cm^2紫外线处理的继代第3－5天的墨西哥黑甜玉米（Zea mays L.cv.Sccharina F.Nigera)胚性悬浮组织原生质体为供体,使用PEG的方法诱导融合。融合再生的18个单细胞克隆的愈伤组织经形态学比较、染色体检查、同工酶分析、5S rRNA间隔序列分析,确认克隆1为杂种愈伤组织,杂种愈伤组织再生出来的白化苗未进行杂种鉴定。同时,初步探讨了紫外线对玉米原生质体的影响。     

同一小麦品种不同来源的两种原生质体与簇毛麦原生质体融合再生杂种植株

小麦(Triticum aestivum L.)济南177的两种原生质体,一种来自快速生长的悬浮细胞,它们因长期继代而丧失分化能力, 其染色体只有2n=24～28;另一种来自可以再生的愈伤组织,其原生质体不能持续分裂.它们中任一种与UV照射过的簇毛麦原生质体融合均不能再生植株.然而当它们混合在一起作为受体时,能够获得再生绿色植株.细胞核基因和胞质基因的分析证明这些绿色植株是杂种.以上事实说明这两种原生质体在融合时存在某些互补的关系,讨论了这种融合方式的可能作用及重要性.     

同一小麦品种不同来源的两种原生质体与簇毛麦原生质体融合再生杂种植株(英文)

小麦 (TriticumaestivumL .)济南 177的两种原生质体 ,一种来自快速生长的悬浮细胞 ,它们因长期继代而丧失分化能力 ,其染色体只有 2n =2 4～ 2 8;另一种来自可以再生的愈伤组织 ,其原生质体不能持续分裂。它们中任一种与UV照射过的簇毛麦原生质体融合均不能再生植株。然而当它们混合在一起作为受体时 ,能够获得再生绿色植株。细胞核基因和胞质基因的分析证明这些绿色植株是杂种。以上事实说明这两种原生质体在融合时存在某些互补的关系 ,讨论了这种融合方式的可能作用及重要性。     

同一小麦品种不同来源的两种原生质与簇毛麦原生质体融合再生杂种植株

小麦（Triticum asetivum L.)济南177的两种原生质体,一种来自快速生长的悬浮细胞,它们因长期继代而丧失分化能力,其染色体只有2n-24-28,另一种来自可以再生的愈伤组织,其原生质体不能持续分裂,它们中任一种与UV照射过的簇毛麦原生质体融合均不能再生植株,然而当它们混合在一起作为受体时,能够获得再生绿色植株,细胞核基因和胞质基因的分析证明这些绿色植株是杂种,以上事实说明这两种原生质体在融合时存在某些互补的关系,讨论了这种融合方式的可能作用及重要性。     

胡萝卜与川西獐牙菜不对称体细胞杂种的核/质基因组特征

胡萝卜(Daucus carota var.sativa)原生质体与经260μW/cm2紫外线照射的川西獐牙菜(Swertia mussotii)原生质体用PEG法诱导融合.对融合再生的克隆的5SrDNA间隔序列和RAPD分析结果得知,各再生克隆均存在双亲的核DNA及重组DNA.杂种的核基因组组成以胡萝卜(受体)为主,供体川西獐牙菜DNA谱带较少;UV照射剂量对体细胞杂种核基因组组成没有明显的影响.进一步对再生克隆叶绿体DNA的SSR分析表明,杂种细胞中双亲叶绿体基因组随机分离并发生重组.     

Regeneration of asymmetric somatic hybrid plants from the fusion of two types of wheat with Russian wildrye

Two types of protoplasts of wheat (Triticum aestivum L. cv. Jinan 177) were used in fusion experiments—cha9, with a high division frequency, and 176, with a high regeneration frequency. The fusion combination of either cha9 or 176 protoplasts with Russian wildrye protoplasts failed to produce regenerated calli. When a mixture of cha9 and 176 protoplasts were fused with those of Russian wildrye, 14 fusion-derived calli were produced, of which seven differentiated into green plants and two differentiated into albinos. The morphology of all hybrid plants strongly resembled that of the parental wheat type. The hybrid nature of the cell lines was confirmed by cytological, isozyme, random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analyses. GISH analysis revealed that only chromosome fragments of Russian wildrye were transferred to the wheat chromosomes of hybrid calli and plants. Simple sequence repeat (SSR) analysis of the chloroplast genome of the hybrids with seven pairs of wheat-specific chloroplast microsatellite primers indicated that all of the cell lines had band patterns identical to wheat. Our results show that highly asymmetric somatic hybrid calli and plants can be produced via symmetric fusion in a triparental fusion system. The dominant effect of two wheat cell lines on the exclusion of Russian wildrye chromosomes is discussed.Abbreviations GISH Genome in situ hybridization - RAPD Random amplified polymorphic DNA - SCF Small chromosome fragment - SSR Simple sequence repeat     

Regeneration of somatic hybrids in relation to the nuclear and cytoplasmic genomes of wheat and Setaria italica.

Somatic hybridization via PEG (Polyethylene 6000)-mediated protoplast fusion was achieved between two different wheat culture lines (Triticum aestivum L., "Jinan"177, T1 and T2) and Setaria italica (L.) P. Beauv. The T1 recipient originated from non-regenerable long-term cell suspensions, while T2 was derived from embryogenic calli with a high regeneration capacity. Donor protoplasts were obtained from embryogenic calli of S. italica (S) (with low regeneration capacity) irradiated with different doses of ultraviolet light. Twenty-three putative hybrid cell lines were produced in fusion combinations with the donor protoplasts treated with UV light for 30 s (combination I) and 1 min (combination II), but only one (from combination II) differentiated into green plants. Three cell lines from combination I and five cell lines from combination II possessed the nuclear genomes of T1, T2, and S. italica as revealed by cytological, isozyme, RAPD, and 5S rDNA spacer sequence analyses. Genomic in situ hybridization (GISH) analysis showed that most hybrid cell lines had 22-36 wheat chromosomes, 0-2 S. italica chromosomes, and 1-6 wheat - S. italica recombinant chromosomes, whereas the regenerable cell line had 44-56 wheat chromosomes and 3-6 recombinant chromosomes, but no intact S. italica chromosomes. RFLP analysis of organellar DNA revealed that mitochondrial and chloroplast DNA of both parents coexisted in all hybrid cell lines and recombined in most hybrid cell lines. These results indicate that the regeneration of hybrid plants involves not only the integration of S. italica nuclear and organellar DNA, but also the genome complementation of T1 and T2.     

Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Common wheat is one of the most important cereal crops in the world. The improvement of its yield and quality by the introduction of heterologous gene(s) is very significant. Avena sativa L. (2n = 42), belonging to the Avena tribe, possesses resistance to drought, coldness and many dis-eases. Its contents of proteins and fat in seed, especially lysine and unsaturated fatty acid are highest in crops, therefore it is regarded as healthy food. Sexual hybridization between wheat and Avena sativa…     

Asymmetric somatic hybridization between wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and <Emphasis Type="Italic">Avena sativa</Emphasis> L.

Protoplasts from cell suspensions of young-embryo-derived calli, whichwere non- regenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of300 μW/cm2 for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

Mixing of maize and wheat genomic DNA by somatic hybridization in regenerated sterile maize plants

Intergeneric somatic hybridization was performed between albino maize (Zea mays L.) protoplasts and mesophyll protoplasts of wheat (Triticum aestivum L.) by polyethylene glycol (PEG) treatments. None of the parental protoplasts were able to produce green plants without fusion. The maize cells regenerated only rudimentary albino plantlets of limited viability, and the wheat mesophyll protoplasts were unable to divide. PEG-mediated fusion treatments resulted in hybrid cells with mixed cytoplasm. Six months after fusion green embryogenic calli were selected as putative hybrids. The first-regenerates were discovered as aborted embryos. Regeneration of intact, green, maize-like plants needed 6 months of further subcultures on hormone-free medium. These plants were sterile, although had both male and female flowers. The cytological analysis of cells from callus tissues and root tips revealed 56 chromosomes, but intact wheat chromosomes were not observed. Using total DNA from hybrid plants, three RAPD primer combinations produced bands resembling the wheat profile. Genomic in situ hybridization (GISH) using total wheat DNA as a probe revealed the presence of wheat DNA islands in the maize chromosomal background. The increased viability and the restored green color were the most-significant new traits as compared to the original maize parent. Other intermediate morphological traits of plants with hybrid origin were not found.     

Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Protoplasts from cell suspensions of young-embryo-derived calli, which were nonregenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of 300 μW/cm² for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

Plant regeneration from intergeneric somatic hybridization between Triticum aestivum L. and Leymus chinensis (Trin.) Tzvel

The suspension derived protoplasts of wheat (Triticum aestivum) cv. Jinan 177 were used as a recipient to fuse with the protoplasts of the ⁶⁰Co gamma-ray irradiated calli of Legmus chinensis. The wheat suspension cells and their protoplasts were not capable of differentiating to whole plants. The irradiated calli of L. chinensis were also the same. The protoplasts originated from the treated or untreated calli were both unable to divide under the conditions of this experiment. However, the fusion products grew and developed to whole plants which were identified as hybrids according to the analysis of chromosome, isozyme and morphology. The above result revealed that the lost regeneration capacity of both parents could be complementarily restored through somatic hybridization. This phenomenon also occurred with our work on Triticum aestivum (+) Haynaldia villosa, T. aestivum (+) Agropyron elongatum and T. aestivum (+) Psathyrostachys juncea.     

Intermediate fertile Triticum aestivum （＋）Agropyron elongatum somatic hybrids are generated by low doses of UV irradiation

We report the production and characterization of somatic hybrids between Triticum aestivum L. and Agropyron elongatum (Host) Nevishi (the synonym is Thinopyrum ponticum). Asymmetric protoplast fusion was performed between Agropyron elongatum protoplasts irradiated with a low UV dose and protoplasts of wheat taken from nonregenerable suspension cultures. More than 40 green plantlets were obtained from 15 regenerated clones and one of them produced seeds. The phenotypes of the hybrid plants and seeds were intermediate between wheat and Agropyron elongatum. All of the regenerated calli and plants were verified as intergeneric hybrids on the basis of morphological observation and analysis of isozyme, cytological, 5SrDNA spacer sequences and random amplified polymorphic DNA (RAPD). RFLP analysis of the mitochondrial genome revealed evidence of random segregation and recombination of mtDNA.