Genome doubling and chromosome elimination with fragment recombination leading to the formation of Brassica rapa-type plants with genomic alterations in crosses with Orychophragmus violaceus.

In distant hybridization of plants, nonclassical hybrids with unexpected chromosome complements, chromosome elimination, and genetic introgression have been well documented. We obtained intergeneric hybrids between Brassica rapa, B. rapa var. chinensis, and another cruciferous species, Orychophragmus violaceus, following embryo rescue. Hybrids mainly displayed phenotypes of B. rapa, although certain O. violaceus or novel characteristics also appeared. Variable numbers of chromosomes were observed in somatic cells in the roots of plantlets on medium and in ovaries and pollen mother cells (PMCs). However, higher numbers were recorded in the roots. GISH revealed that the majority of ovary cells and PMCs contained 20 chromosomes of B. rapa with or without individual O. violaceus chromosomes or fragments added or introgressed. AFLP analysis showed that fragments deleted from the B. rapa genome were much more frequent than novel and O. violaceus fragments. The mechanisms involved genome doubling and successive elimination of O. violaceus chromosomes accompanied by fragment recombination and introgression, producing B. rapa-type plants with modified genetic constitutions and phenotypes.     

Cytogenetic and molecular characterization of intergeneric hybrids between Brassica napus and Orychophragmus violaceus.

Twenty-two intergeneric hybrids from a cross between Brassica napus (AACC, 2n = 38) cultivar Oro and the ornamental crucifer Orychophragmus violaceus (OO, 2n = 24) were produced without embryo rescue. The plants were classified into three groups based on morphological and cytological observations and RAPD banding patterns. Plants of Group I had morphological traits of both parents and 2n = 29 chromosomes. In these plants, 62.1% of the pollen mother cells (PMCs) had the pairing configuration 1 III + 9 II + 8 I; the remaining PMCs had 10 II + 9 I. The plants possessed 97.6-98.8% B. napus specific and 9.2-11.7% O. violaceus specific RAPD fragments. Plants of Group II exhibited novel morphological traits and possessed 2n = 35, 36, or 37 chromosomes. Plants of Group III were morphologically similar to B. napus and possessed 2n = 19, 37, 38, or 39 chromosomes. Plants of Group II and Group III had 94.1-99.4% B. napus specific RAPD fragments and no O. violaceus specific RAPD fragments. Chromosome fragments were observed in PMCs of most of the F1 plants in all groups. Based on the cytological results and RAPD analysis, it is suggested that genome doubling and chromosome elimination occurred in the intergeneric hybrids of B. napus x O. violaceus.     

Phosphinothricin-resistant somatic hybrids Brassica napus + Orychophragmus violaceus

Phosphinothricin (PPT) resistant hybrid plants between Brassica napus L. cv. Kalinovsky and Orychophragmus violaceus (L.) O.E. Shulz. were obtained as a result of somatic hybridization experiments. The hybrids inherited PPT resistance from O. violaceus plants which were previously transformed by the vector containing Spm/dSpm Zea mays transposon system with bar gene located within the nonautonomous transposon. The obtained plants had intermediate morphology. Their hybrid nature has been confirmed by isozyme (esterase and amilase activity) and PCR (bar, gus, Spm/dSpm integration) analyses. The hybrids combined B. napus plastom and O. violaceus mithochondrion that was revealed by PCR-RFLP. The hybrid plants might be included to rapeseed breeding programme after examination of their oil quality as well as to chloroplast transformation experiments that is still urgent for B. napus.     

Alteration of chromosome behavior and synchronization of parental chromosomes after successive generations in Brassica napus x Orychophragmus violaceus hybrids.

In an earlier study, the progenies of intergeneric hybrids Brassica napus (2n = 38) x Orychophragmus violaceus (2n = 24) were investigated in successive generations (F1-F4) for the cytological phenomenon of parental genome separation during mitotic and meiotic division. In the present study, inbred lines (F5-F8) derived from 1 such hybrid were characterized for morphology, chromosome pairing behaviour, and genome composition. One F5 plant (2n = 31) with slightly yellow petals and 12:19 and 15:16 segregation ratios in its pollen mother cells (PMCs) produced F6 plants with distinct morphological characteristics and wide variations in fertility and chromosome numbers (2n = 25-38). F7 and F8 lines with distinctive morphology and wide ranges in chromsome numbers were established. In PMCs of F7 plants from 4 F6 plants, 0-12 labelled chromosomes from O. violaceus, which predominantly appeared as bivalents, were identified by genomic in situ hybridization. They behaved synchronously with B. napus chromosomes during meiotic division. The results provide molecular cytogenetic evidence of the inclusion of O. violaceus chromosomes in the original hybrids and the cytology in the hybrids documented earlier. They also show that chromosome behaviour was altered and the parental chromosomes became synchronized after successive generations.     

Parental genome separation and elimination of cells and chromosomes revealed by AFLP and GISH analyses in a Brassica carinata x Orychophragmus violaceus cross

BACKGROUND AND AIMS: The phenomenon of parental genome separation during the mitotic divisions of hybrid cells was proposed to occur under genetic control in intergeneric hybrids between cultivated Brassica species and Orychophragmus violaceus (2n = 24). To elucidate further the cytological and molecular mechanisms behind parental genome separation, Brassica carinata (2n = 34) x O. violaceus hybrids were resynthesized and their chromosome/genomic complements analysed. METHODS: F(1) hybrids of the cross were obtained following embryo rescue, and were investigated for their cytological behaviour and subjected to genomic in situ hybridization (GISH) and amplified fragment length polymorphism (AFLP) to determine the contribution of parental genomes. KEY RESULTS: All the F(1) plants with high fertility closely resembled B. carinata in morphological attributes. These were mixoploids with 2n chromosome numbers ranging from 17 to 35; however, 34, the same number as in B. carinata, was the most frequent number of chromosomes in ovary and pollen mother cells (PMCs). GISH clearly identified 16 chromosomes of B. nigra in ovary cells and PMCs with 2n = 34 and 35. However, no O. violaceus chromosome was detected, indicating the presence of the intact B. carinata genome and elimination of the entire O. violaceus genome. However, some AFLP bands specific for O. violaceus and novel for the two parents were detected in the leaves. Cells with fewer than 34 chromosomes had lost some B. oleracea chromosomes. F(2) plants were predominantly like B. carinata, but some contained O. violaceus characters. CONCLUSIONS: The cytological mechanism for the results involves complete and partial genome separation at mitosis in embryos of F(1) plants followed by chromosome doubling, elimination of cells with O. violaceus chromosomes and some introgression of O. violaceus genetic information.     

Intergeneric hybrids between Brassica napus and Orychophragmus violaceus containing traits of agronomic importance for oilseed rape breeding

Protoplast fusions between Brassica napus and Orychophragmus violaceus for transfer of valuable traits to oilseed rape resulted in 257 somatic hybrid plants. Hybridity was confirmed by morphological, cytological and molecular means. Symmetric fusions gave rise to 131 plants. Fifty eight of these plants had an intermediate morphology and contained nuclear DNA corresponding to the sum of the parental species. All 131 plants were sterile with no pollen grains observed upon flowering. Another 126 plants were derived from asymmetric fusions in which protoplasts of the donor parent O. violaceus were irradiated by 100 or 200-Gy X-rays prior to fusion. Morphologically these plants showed a larger variation compared to the plants regenerated from symmetric fusion experiments. In contrast to plants obtained from symmetric fusions, fertile hybrids were recovered among regenerants from the asymmetric fusions. Twenty four of these plants released viable pollen grains and 14 of the determined 17 plants set seeds after either selfing or backcrossing to B. napus. Fourteen male-sterile plants were identified with female fertility. This observed male sterility most-likely originated from alloplasmic recombination and would be of great potential for the development of a new cytoplasmic male sterility system. The fatty acid composition of the fertile hybrids and their progenies showed a biased distribution towards the B. napus parent, which has a high erucic acid-content type. However, increased levels of palmitic and linoleic acids compared to B. napus were found in subsequent generations, as well as a reduced level of erucic acid.     

Regeneration and characterisation of Cucumis melo L. (+) Cucumis anguria L. var. longipes (Hook. fil.) Meeuse somatic hybrids

Cotyledon protoplasts of an albino Cucumis melo L. ‘Cantaloup Charentais’ somaclonal variant were electrofused with protoplasts of the wild species Cucumis anguria L. var. longipes (Hook. fil.) Meeuse. Selection of putative somatic hybrids was based on competence of the albino melon to grow and regenerate shoots together with the ability of the wild species to synthesise chlorophyll. The ITS region of C. anguria ribosomal DNA was sequenced to design species-specific primers, which allowed us to distinguish between parental lines and fusion products by PCR amplification. By using this method, all the organogenic lines characterised proved to be somatic hybrids. Three of sixteen selected lines produced shoots with albino and green sectors. Eleven lines remained green but shoots developed abnormally and did not produce roots in vitro. Two hybrid lines regenerated normal shoots but with a limited ability to produce roots in vitro and in vivo. Applicability of molecular characterisation to optimise the quick recovery of fusion products is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phosphinothricin-resistant <Emphasis Type="Italic">Brassica napus</Emphasis> + <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> somatic hybrids

Hybrid plants resistant to phosphinothricin (PPT) are obtained as a result of experiments with somatic hybridization between Brassica napus L. cv. Kalinins’kyy and Orychophragmus violaceus L. O.E. Shulz. The hybrids inherited PPT resistance from O. violaceus plants that had been previously transformed by a vector containing the maize transposon system Spm/dSPm with bar gene located within the nonautonomous transposon. The morphologically obtained plants occupy an intermediate position between the initial forms, which is in agreement with the results of isoenzyme analyses (analysis of multiple forms of amylase and esterase) and PCR analysis (presence of the genes bar, gus, and SpmTPase). Inheritance of the plastome occurs from oilseed rape, while that of the mitochondrion, from O. violaceus, which is proved by means of PCR-RFLP analysis. The plant hybrids may be utilized for further selection research with oilseed rape following determination of the edible quality of its oil as well as in experiments with chloroplast transformation, a topic which is of critical importance for oilseed rape.     

Activity of the corn Spm transposon system in transgenic plants Orychophragmus violaceus (L.) O.E. Schulz obtained by both direct transfer of DNA to protoplasts and agrobacterial transformation of root explants

Transposon mediated insertional mutagenesis is one of the approaches for the unique gene cloning. A wild species of Cruciferae family Orychophragmus violaceus (L.) O.E. Schulz, which is of interest for practical breeding as a donor of improved plant oil, was an object of the investigation. Plasmid construction used in the experiments included selective NPT II gene, reported GUS gene serving as an excision marker, structural BAR gene located within the dSpm element and Spm transposase. The GUS gene of this plasmid had not his own promoter and became functional only after Spm-transposition. Transformed Orychophragmus violaceus (L.) O.E. Schulz. plants were obtained by direct mesophyll protoplast transformation as well as Agrobacterium tumefaciens-mediated root explant transformation. Gene transfer and the transposition event were confirmed by the GUS activity and the PCR analysis. Relative transformation efficiency using protoplasts was 5.8%.     

Callus Induction and Plant Regeneration of Leaf Explants in Orychophragmus violaccus(L.) O. E.Schulz

Explants of Orychophragmus violaceus (L.) O. E. Schulz were acquired from young leaves which lower epidermis was stripped, Differentiation of calli in high frequency is in the case of that calli grown on B5 culture medium supplemented with 1 mg/l 2, 4-D should be transferred onto MS culture medium supplemented with 0.2 mg/l NAA. Effect of basic culture medium on the differentiation was discussed. In addition, protoplasts derived from calli of Orychophragmus violaceus were cultured, and small calli consisted of more than hundred cells had been obtained.     

Limited DNA elimination from the irradiated potato parent in fusion products of albino Lycopersicon esculentum and Solanum tuberosum

Summary This paper describes the analysis of the elimination of potato DNA from potato-tomato somatic cell hybrids. The hybrids were obtained by fusion of protoplasts of a cytoplasmic albino tomato genotype with leaf mesophyll protoplasts of a potato genotype carrying the -glucuronidase (GUS) gene of Escherichia coli. The potato protoplasts were either isolated from unirradiated plants or from plants irradiated with 50 or 500 Gy of -rays. Green calli were selected as putative fusion products. The hybridity of these calli was confirmed by isoenzyme analysis. All of the green calli tested contained a potato-specific chloroplast DNA restriction fragment, and most of the calli analysed were positive for -glucuronidase activity. In 72 of the hybrid calli we determined the percentage of potato nuclear DNA using species-specific probes. All of the tested green calli contained a considerable amount of potato genomic DNA, irrespective of the dose of irradiation of the potato parent. The limited degree of potato DNA elimination and the absence of true cybrids are discussed.     

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.     

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.     

中国特有诸葛菜复合群的系统发育关系

利用5．8S核糖体DNA全长间隔序列（ITS／5．8S）和叶绿体基因mat K对中国原产诸葛菜复合群的系统发育关系进行了分析研究。ITS序列结果支持中国诸葛菜复合群分为两支：一支由湖北诸葛菜和太白诸葛菜组成;另一支由诸葛菜和铺散诸葛菜组成。mat K的序列分析结果表明,在中国诸葛菜复合群的系统发育树中,铺散诸葛菜和诸葛菜处于基部位置。结合以前的核型分析和本文的研究结果,我们不支持将湖北诸葛菜和太白诸葛菜归隶于诸葛菜,而支持我们之前提出的两种可能的原始细胞型的进化假说。另外,根据生物地理学分析,推测从长江中下游到秦岭地区是中国原产诸葛菜的近代分化中心。     

甘蓝型油菜与诸葛菜属间杂种的减数分裂观察

在甘蓝型油莱与诸葛菜属间杂种细胞减数分裂中,观察到两种类型的染色体分离方式。第一种,细胞内的３１条染色体呈两组分离,其中１９条染色体至细胞一极,１２条染色体至另一极;且在两极的染色体进行分裂,产生４个核,两个具有１９条染色体,另两个具有１２条染色体。故杂种细胞通过这种染色体分离方式,可同时产生具有１９与１２条染色体的两类配子。杂种细胞内发生的这种染色体行为,极有可能与来自两个亲本种的染色体组相关;即在细胞两极的１９与１２条染色体分别来自甘蓝型油菜与诸葛菜。第二种,在后期Ｉ至末期Ｉ,花粉母细胞内以６个二价体形式出现的１２条染色体被落后在赤道板附近,而被排斥在末期核之外。这些落后二价体很有可能来自诸葛菜。因此,在杂种细胞减数分裂中,来自两个亲本种的染色体组被分开。文中还对染色体组分开的遗传意义等进行了讨论。     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Construction of a primary RH panel of Italian ryegrass genome via UV-induced protoplast fusion

Symmetric and asymmetric somatic hybrids were produced via protoplast fusion between common wheat ( TRITICUM AESTIVUM L.) cv. "Jinan 177" and Italian ryegrass ( LOLIUM MULTIFLORUM Lam.). The ryegrass without or with UV irradiation was used as a donor, providing a small amount of chromatin. In these somatic hybrids, most ryegrass chromosomes have been confirmed preferential elimination and the somatic hybrid calli and plants showed wheat-like morphology. Some of the hybrid lines were used for the analysis of distribution and heredity of donor DNA in the hybrid genome and the possibility of establishing a radiation hybrid (RH) panel of the ryegrass in the present experiment. These hybrids, subcultured for two and three years, retained the ryegrass DNA examined by RFLP and GISH analysis, respectively. Distribution of the ryegrass DNA in the wheat genomes of 20 single-cell individuals, randomly selected from hybrid cell lines produced, were analyzed by 21 ryegrass genome specific SSR markers. The average frequencies of molecular marker retention in symmetric hybrid lines (UV 0), as well as asymmetric hybrid lines from UV 30 s and 1 min were 10.88, 15.48 and 33.86, respectively. It was suggested that the UV dose increased the introgression of donor DNA into wheat genome. The ryegrass SSR fragments in most asymmetric hybrid cell lines remained stable over a period of 2 approximately 3 years. This revealed that those asymmetric somatic hybrids are suitable for the introgression of ryegrass DNA into wheat, and for RH panel and RH mapping.     

不同植物的碳酸酐酶活力差异研究

碳酸酐酶是催化二氧化碳的可逆水合反应的一种含锌金属酶。测定不同植物、同一植物不同部位、同一植物同一部位不同时间的碳酸酐酶的活力,研究诸葛菜和油菜碳酸酐酶及其胞外酶活力的差异,初步探讨碳酸酐酶活力与植物抗干旱能力之间的关系。研究结果为诸葛菜的喀斯特适生性的研究提供依据。     

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     

Production of somatic hybrids between Diospyros glandulosa and D. kaki by protoplast fusion

Interspecific somatic hybrids between Diospyros glandulosa (2n=2x=30) and D. kaki cv. Jiro (2n=6x=90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using a modified KM8p medium. Flow cytometry revealed that the nuclear DNA content was the sum of those of D. glandulosa and D. kaki cv. Jiro in 149 of the 166 calli obtained. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and further confirmed that they were interspecific somatic hybrid calluses. Shoots were regenerated from 63 of the 149 interspecific hybrid calluses. Chloroplast DNA analysis by PCR-RFLP, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained the nuclear genomes from both parents but only the chloroplast genome from D. glandulosa. Microscopic observation of root tip cells demonstrated that somatic chromosome number of the interspecific hybrids was 2n=8x=120. This revised version was published online in June 2006 with corrections to the Cover Date.