Analysis of chromosomal and organellar DNA of somatic hybrids between Triticum aestiuvm and Haynaldia villosa Schur

Intergeneric somatic hybridization between wheat (cv. Jinan 177) protoplasts that have 24-28 chromosomes and Haynaldia villosa protoplasts containing 11-14 chromosomes was carried out by the polyethylene glycol (PEG) method. A high frequency of hybrid calli and plants were obtained from the fusion products, as revealed by cytological and biochemical techniques and by PCR analysis of 5S rDNA spacer sequences. GISH (genomic in situ hybridization) analysis confirmed the presence of chromosomes from both parents in the hybrid clones and the common occurrence of translocations between them. The RFLP analysis of the organellar DNA using mitochondrion- and chloroplast-specific probes revealed that mitochondria from both parents existed in the cells of hybrid calli and their recombination, whereas chloroplasts segregated and recombined randomly. The gross morphology of hybrid plants resembled that of wheat, but the gross morphology of their ovaries and anthers were intermediate between those of the two parents. The relationship between hybrid plant regeneration and the balance of genetic materials in hybrid clones is discussed.     

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     

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.     

通过不对称体细胞杂交向小麦转移青苗碱谷DNA

普通小麦"济南177"(Triticum aestivum L.cv.Jinan177)经继代培养和选择,形成了性质不同的愈伤组织,一种生长迅速,易于形成悬浮系,游离的原生质具有旺盛的分裂能力,但不能分化,称为Cha9;另一种具有一定能力,但游离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.而将源于Cha9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现型、细胞学、有工酶、RAPD分析,证明了其杂种性质,用不麦叶绿体特异的简单得复序列(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Cha9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.     

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.     

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。     

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…     

Somatic embryogenesis and plant regeneration from protoplasts isolated from embryogenic cell suspensions of wheat (Triticum aestivum L.)

We describe the early formation of somatic embryos followed by plant regeneration from protoplasts isolated from an embryogenic wheat cell suspension, which was initiated from small granular (0.2 to 1 mm in size) embryogenic calli. These granular calli formed embryogenic cell suspensions within 20 days in liquid culture, and were selected gradually from young inflorescence-derived nodular embryogenic calli of the winter wheat cv. Kehong 1041. The division frequency of protoplasts was 11 to 16%, and the frequency of differentiation into plants was about 0.001% (number of plants formed divided by the total number of protoplasts plated). About 20% of somatic embryos present in the culture formed directly from protoplast-derived cells within 15 days of cultures.     

通过“供体-受体”原生质体融合将裸燕麦部分核基因组转移给普通小麦(英文)

本文进行了小麦和裸燕麦悬浮细胞原生质体的电融合,并基于双亲失活(用IOA处理受体小麦原生质体,用γ-射线照射供体裸燕麦细胞系),获得可能的杂种愈伤组织。对7块愈伤组织进行了乙醇脱氢酶(Adh)同工酶筛选,发现5块表现出双亲特征酶带。对3个杂种细胞系进行5种同工酶分析,证实它们均为稳定的不对称体细胞核杂种细胞系;它们表现出小麦的完整谱带和裸燕麦的部分谱带。对2个杂种细胞系及亲本的核糖体DNA Southern分析结果表明只有一个杂种细胞系(HB 95)含有双亲的全部谱带。细胞学观察表明,2个杂种细胞系的染色体数目均显著高于双亲。从杂种细胞系HB 94中分化出叶原基等分化结构。Adh同工酶分析表明,这些分化结构具有和母体细胞系完全相同的杂种谱带。     

Callus Formation from Somatic Hybridization of Wheat (Triticum aestivum L.) and Ryegrass (Lolium perenne L.) by Electrofusion

Somatic hybrid calli were recovered following electrofusion of protoplasts from a chloroplast-containing cell suspension culture of wheat (Triticum aestivum L.) and a cell suspension culture of ryegrass (Lolium perenne L.). The protoplasts of wheat were inactivated by iodoacetamide; in addition morphology and colour were used as markers to aid selection of putative hybrid calli. For isozyme analysis of putative hybrids, nine isozymes were tested for differences in bands between the parental lines. Of these, three showed differences (ADH, GOT, SDH). Analysis of ADH bands of calli indicated that six lines were hybrids. These lines were analysed with the ,ther isozymes, and at the DNA level by Southern hybridisation with a wheat ribosomal DNA probe. The overall results indicated that one line was an almost complete combination of the genomes of the parental lines, but the other 5 lines were probably partial hybrids. In the latter, some loss of the wheat genome had probably occurred.     

小麦与谷子不对称体细胞杂交

以不同剂量的紫外线(UV)照射谷子(Setariaitalica cy. Shanxi)原生质体为供体与普通小麦(Triticum aestivum)济南177和99P的原生质体用PEG法诱导融合.利用同工酶,RAPD和染色体分析再生的克隆及白化苗.从小麦济南177与谷子融合再生的86个克隆中,24个被鉴定为杂种克隆;而小麦99P与谷子融合再生的67个克隆中,27个被鉴定为杂种克隆.虽然用作融合材料的双亲培养细胞均丧失再生能力,但是部分来源于低剂量UV处理的杂种克隆再生了绿点、根和白化苗.证明小麦体细胞杂交中的双亲再生能力互补现象也存在于远缘族间融合组合中.讨论了小麦远缘族间融合未能再生绿色植株的原因.     

The effect of rye chromosomes on callus induction and regeneration in callus cultures of immature embryos of wheat-rye substitution lines, Triticum aestivum L. cultivar Saratovskaia 29/Secale cereale L. cultivar Onokhoiskaia

The effect of individual rye chromosomes on the induction of callus and the character of its regenerating capacity was studied with cultured immature embryos of wheat-rye (Triticum aestivum L. cv. Saratovskaya 29-Secale cereale L. cv. Onokhoiskaya) substitution lines. The genotypic diversity of the substitution lines proved to significantly affect variation of parameters characterizing the major types of callus cultures, that is, frequencies of embryogenic calli, which are capable of shoot regeneration, and of morphogenic calli, which produce root structures. Functioning in the genotypic background of common wheat cultivar Saratovskaya, chromosomes 2R and 3R of rye cultivar Onokhoiskaya stimulated significantly the induction of embryogenic callus highly capable of shoot regeneration. Rye chromosome 2R present in place of chromosome 2D in the common wheat genome suppressed the induction of callus producing root structures. Rye chromosomes 1R and 6R suppressed the induction of embryogenic callus capable of shoot regeneration.     

小麦胚性悬浮系与原生质体植株再生

普通小麦(Triticum aestivum)昌乐5号(冬性)胚性悬浮细胞系的组成成分对原生质体再生频率发生影响,此种悬浮系是由混合型愈伤组织建立起来的,建成的悬浮系中含有2—3mm的小愈伤组织和分散好的几十至上百个细胞的细胞团。分别用悬浮系中的小愈伤组织和细胞团分离原生质体进行培养。结果表明．由小愈伤组织来源的原生质体再生植株的频率显著高于由细胞团分离的原生质体的再生频率。培养基中不同成分对原生质体分裂的影响也作丁研究。     

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.     

The Effect of Rye Chromosomes on Callus Induction and Regeneration in Callus Cultures of Immature Embryos of Wheat–Rye Substitution Lines,Triticum aestivum L. Cultivar Saratovskaya 29–Secale cereale L. Cultivar Onokhoiskaya

The effect of individual rye chromosomes on the induction of callus and the character of its regenerating capacity was studied with cultured immature embryos of wheat–rye (Triticum aestivum L. cv. Saratovskaya 29–Secale cereale L. cv. Onokhoiskaya) substitution lines. The genotypic diversity of the substitution lines proved to significantly affect variation of parameters characterizing the major types of callus cultures, that is, frequencies of embryogenic calli, which are capable of shoot regeneration, and of morphogenic calli, which produce root structures. Functioning in the genotypic background of common wheat cultivar Saratovskaya 29, chromosomes 2R and 3R of rye cultivar Onokhoiskaya stimulated significantly the induction of embryogenic callus highly capable of shoot regeneration. Rye chromosome 2R present in place of chromosome 2D in the common wheat genome suppressed the induction of callus producing root structures. Rye chromosomes 1R and 6R suppressed the induction of embryogenic callus capable of shoot regeneration.     

The chromosome content and genotype of two wheat cell lines and of their somatic fusion product with oat

Somatic hybridization seeks to genetically combine phylogenetically distant parents. An effective system has been established in bread wheat (Triticum aestivum L.) involving protoplasts from a non-totipotent cell line adapted to in vitro culture (T₁) in combination with totipotent protoplasts harvested from embryogenic calli (T₂). Here, we report the karyotype and genotype of T₁ and T₂. Line T₁ carries nine A (A-genome of wheat), seven B (B-genome of wheat) and eight D (D-genome of wheat) genome chromosomes, while T₂ cells have 12 A, 10 B and 12 D genome chromosomes. Rates of chromosome aberration in the B- and D-genomes were more than 25%, higher than in the A-genome. DNA deletion rates were 55.6% in T₁ and 19.4% in T₂, and DNA variation rates were 8.3% in T₁ and 13.9% in T₂. Rate of DNA elimination was B- > D- > A-genome in both T₁ and T₂. The same set of cytological and genetic assays was applied to a derivative of the somatic fusion between protoplasts of T₁, T₂ and oat (Avena sativa L.). The regenerant plants were near euploid with respect to their wheat complement. Six wheat chromosome arms—4AL, 3BS, 4BL, 3DS, 6DL and 7DL—carried small introgressed segments of oat chromatin. A genotypic analysis of the hybrid largely confirmed this cytologically-based diagnosis.     

小麦与多年生黑麦草原生质体的电融合及杂种愈伤组织的形成

多年生黑麦草(Lolium perenne L.)悬浮培养细胞来源的原生质体和小麦(Triticumaestivum L.)含叶绿体的悬浮培养细胞来源的原生质体间,用直流方波脉冲进行电融合,获得了体细胞杂种愈伤组织。小麦的原生质体经过碘乙酰胺失活。愈伤组织的形态和颜色被用作识别预期杂种的标记。为了对杂种愈伤组织进行同工酶分析,观察了亲本的9种同工酶谱,其中3种在亲本间表现出差异(ADH、GOT 和 SDH)。酒精脱氢酶(ADH)的分析结果表明,有6个细胞系表现出杂种带。这些细胞系经过其他两种同工酶分析和 rDNA 探针杂交试验表明,一个细胞系表现出基本完全的亲本基因组间的组合,其余5个细胞系是部分杂种。     

Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis

Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.     

The somatic embryogenesis and establishment of transformation experiment system in Larix principis-Rupprechtii]

Larix principis-Rupprechtii is one of the superior afforestation forest trees growing in north China. Embryogenic cultures were initiated from immature zygotic embryos of Larix principis-Rupprechtii on S culture medium containing 2, 4-D 0-2.2 mg/L, KT and BA each at 0-0. 8 mg/L. Embryogenic calli were subcultured and multiplicated on S + B culture medium containing dropping off each hormone concentration. We set up 33 steady-going embryogenic cell lines; We studied on the growth stage and genotype differences of every embryogenic cell lines; and Finded more than 10 high-frequency somatic embryogenesis cell lines such as 2K, 2T, 2I, 2J, 3C etc.. The number of 2T somatic embryos reaches 314/per gram of embryogenic tissue and the number of 3C somatic embryos is 185/per gram of embryogenic tissue. The re-induction method of Larix principis-Rupprechtii from somatic embryos was used to produce renewable embryogenic cultures and steady-going embryogenic cell lines effectively. Mature somatic embryos can germinate and develop further into plantlets when they are isolated and cultured on a hormone-free WPM culture medium. The regeneration plantlets were obtained. Furthermore, the transformation with a truncated gene of Bacillus thuringensis (B. t) were carried out, the PCR showed positive results, because of this, embryogenic cell line of Larix principis-Rupprechtii can be used for transformation experiments to support further breeding in forestry.     

Nuclear genomic composition of asymmetric fusion products between irradiated transgenic Solanum brevidens and S. tuberosum: limited elimination of donor chromosomes and polyploidization of the recipient genome

Summary The production of asymmetric somatic hybrid calli after fusion between gamma-irradiated protoplasts from transgenic Solanum brevidens and protoplasts from S. tuberosum are reported. Transgenic (kanamycin-resistant, GUS-positive) S. brevidens plants and hairy root clones were obtained after transformation with Agrobacterium tumefaciens LBA 1060 (pRi1855) (pBI121) and LBA 4404 (pRAL4404) (pBI121), and A. rhizogenes LBA 9402 (pRi1855) (pBI121), respectively. Leaf protoplasts isolated from the transgenic plants or root protoplasts from the hairy root clones were fused with S. tuberosum leaf protoplasts, and several calli were selected on kanamycin-containing medium. The relative nuclear DNA content of the hybrid calli was measured by flow cytometry (FCM), and the percentages of DNA of the S. brevidens and S. tuberosum genomes in the calli were determined by dot blot analysis using species-specific DNA probes. Chromosome-specific restriction fragment length polymorphism (RFLP) markers were used to investigate the elimination of specific S. brevidens chromosomes in the hybrids. The combined data on FCM, dot blot and RFLP analysis revealed that 18–62% of the S. brevidens DNA was eliminated in the hybrid calli and that the RFLP marker for chromosome 7 was absent in seven out of ten calli. The absence of RFLP markers for chromosomes 5 and 11 hardly ever occurred. In most of the hybrids the ploidy level of the S. tuberosum genome had increased considerably.