Production and cytogenetics of intergeneric hybrids between Brassica napus and Orychophragmus violaceus

The intergeneric hybrid between Brassica napus and Orychophragmus violaceus was obtained by means of embryo culture technique with the latter as the pollen parent. The hybrid was morphologically intermediate between its parents, but could produce a lot of seeds when selfed. Somatic separation of the genomes from the two parental species was observed during the mitotic divisions of some of the hybrid cells. Thus, the hybrid became the mixoploid in nature, consisting of haploid and diploid cells of B. napus, and a nuclear — cytoplasmic hybrid, with the cytoplasm of B. napus and the nuclei of O. violaceus, and the hybrid cells. Pollen mother cells with 19, 12 and 6 bivalents, respectively, were produced by the hybrid. From the selfed progeny of the hybrid, mainly two kinds of plants, B. napus and the hybrid, were found. The hybrid plants of the selfed progeny again produced two kinds of plants, B. napus and the hybrid.     

Production and cytogenetics of the intergeneric hybrids Brassica juncea×Orychophragmus violaceus and B. carinata×O. violaceus

 Intergeneric hybrids between Brassica juncea (2n=36), B. carinata (2n=34) and Orychophragmus violaceus (2n=24) were produced when B. juncea and B. carinata cultivars were used as female parents. The hybrids between B. juncea and O. violaceus had an intermediate morphology except for petal colour and were partially fertile. The hybrids between B. carinata and O. violaceus had a matroclinous morphology and were nearly fertile. Cytological analysis of the hybrids and their progenies gave the following results. (1) In the hybrids between B. juncea and O. violaceus, the somatic tissues of the roots, leaves and styles were mixoploid (2n=12–42), and cells with 24, 30 or 36 chromosomes were the most frequent. Based on the recorded numbers and behaviour of the mitotic and meiotic chromosomes, complete and partial separation of the parental genomes was proposed to have occurred during mitosis. This resulted in the occurrence of cells with possibly complete and incomplete complements of the parental species and cells with parental complements and some additional chromosomes from the other parent. (2)  Pollen mother cells (PMCs) possibly with both parental chromosome complements, only B. juncea chromosomes or a complete B. juncea complement with additional O. violaceus chromosomes were more competitive in entering meiosis. The majority of fertile gametes were deduced to have been produced by PMCs with a B. juncea complement with or without additional O. violaceus chromosomes. (3) The progeny plants from selfed hybrids between B. juncea and O. violaceus were morphologically either of a B. juncea, hybrid or variable type. Cytologically they were grouped into six types according to the frequencies of cells with various chromosome numbers. All of the plants except 2 which constituted two types, were mixoploids, composed of cells with various chromosome numbers, mainly in a certain serial range. (4) The hybrid plants between B. carinata and O. violaceus were mixoploids with chromosome numbers in the range of 12–34, and cells with 2n=34 were the most frequent. The main categories of PMCs with 17 bivalents at metaphase I and 17 : 17 segregations at anaphase I contributed to the high fertility of the hybrids and the fact that their progeny after selfing were mainly plants with 2n=34. Somatic and meiotic separation of the parental genomes was proposed to have occurred in the hybrids between B. carinata and O. violaceus. (5) Mitotic and meiotic elimination of what could be O. violaceus chromosomes might also have contributed to the observed mitotic and meiotic cell types in the two kinds of hybrids studied. Finally, the possible mechanisms behind these cytological observations and their potential in the production of Brassica aneuploids were discussed. Received: 4 February 1997/Accepted: 29 July 1997     

甘蓝型油菜与诸葛菜属间杂种无性系的变异研究

从形态及细胞学两方面对甘蓝型油菜与诸葛菜属间杂种无性系的变异进行了研究。经过长期继代培养后,杂种在形态上越来越偏向母本甘蓝型油菜,而表现出较少的父本诸葛菜性状,但表现出对等双分枝的新性状。经细胞学观察表明,杂种体内杂种细胞比例下降,而甘蓝型油菜细胞比例大幅度上升并远高于核质杂种细胞（具有油菜细胞质与诸葛菜细胞核）的比例,以至较多的甘蓝型油菜染色体组被遗传下去。在该杂种继代培养中还观察到杂种细胞内的染色体消除及体细胞配对现象。     

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。     

Production and characterization of interspecific hybrids between Brassica maurorum and crop brassicas

 Seven interspecific hybrids were produced between Brassica maurorum (♀), a wild species resistant to Alternaria blight and white rust, and all the monogenomic (B. campestris, B. nigra and B. oleracea) and digenomic (B. juncea, B. napus and B. oleracea) crop brassicas (♂) through embryo rescue. The hybrids were confirmed by means of morphological and cytological studies. All the hybrids were pollen-sterile. Amphidiploids were induced in three of the hybrids: B. maurorum×B. napus, B. maurorum×B. carinata, B. maurorum×B. nigra. The hybrids were also confirmed through DNA analyses for nuclear and organelle genomes using RAPD and RFLP techniques. Received: 31 July 1998 / Accepted: 14 August 1998     

植物逆转座子及其在基因功能和基因组分析中的应用

植物逆转座子及其在基因功能和基因组分析中的应用路铁刚孙敬三（中国科学院植物研究所,北京１０００９３）ＰｌａｎｔＲｅｔｒｏｔｒａｎｓｐｏｓｏｎｓａｎｄｔｈｅＰｅｒｓｐｅｃｔｉｖｅｏｆＴｈｅｉｒＡｐｐｌｉｃａｔｉｏｎｉｎＧｅｎｅＦｕｎｃｔｉｏｎａｌａｎｄ...     

Camalexin induction in intertribal somatic hybrids between Camelina sativa and rapid-cycling Brassica oleracea

  Camelina sativa, a wild relative of Brassica crops, is virtually immune to blackspot disease caused by Alternaria brassicicola. Intertribal somatic hybrids were produced between C. sativa and rapid-cycling Brassica oleracea as a step toward the transfer of resistance to this disease into Brassica vegetable crops. The plants recovered were confirmed as somatic hybrids by flow cytometry and RAPD analysis. All hybrids showed a morphology intermediate between the two parents. Rooted plants grew in soil up to 4–5 weeks, and some produced sterile flowers. Two of three hybrids tested showed a high level of resistance to  A. brassicicola. Resistance was correlated with the induction of high levels of the phytoalexin camalexin 48 h after inoculation, as in the resistant Camelina fusion partner. In contrast, susceptible somatic hybrids produced much lower levels of camalexin. Received: 2 April 1998 / Accepted: 14 July 1998     

Production and cytogenetics of Brassica campestris-alboglabra chromosome addition lines

 Four different Brassica campestris-alboglabra monosomic addition lines (AA+1 chromosome from C, 2n=21) were obtained after consecutive backcrosses between resynthesized B. napus (AACC, 2n=38) and the parental B. campestris (AA, 2n=20) accession. The alien chromosomes of B. alboglabra (CC, 2n=18) in the addition lines were distinguished by random amplified polymorphic DNA (RAPD) marker analysis and morphology of mitotic chromosomes. Four RAPD marker synteny groups were established, which represented the four different alien chromosomes of B. alboglabra in the four addition lines. Three of the four addition lines were identified to harbour chromosomes 4, 8 or 9 of B. alboglabra. Studies on meiotic pairing in the addition lines revealed intergenomic homoeology relationships among specific chromosome arms between the A- and C-genomes. The long arm of B. campestris chromosome 9 was homoeologous with the long arm of B. alboglabra chromosome 4, while its short arm with the short arms of B. alboglabra chromosomes 8 and 9. Such an intergenomic homoeology relationship supports the hypothesis that B. campestris and B. alboglabra share a common ancestor but that chromosomal rearrangements have occurred during the evolution of the two species. Intergenomic introgression was observed in the progenies of the addition lines. The introgression of an entire B. alboglabra marker synteny group into the B. campestris genome implied the possible occurrence of interspecific chromosomal substitution. Received: 30 May 1996 / Accepted: 18 October 1996     

Production and characterization of intergeneric somatic hybrids of Trachystoma ballii and Brassica juncea

Intergeneric somatic hybrids, Trachystoma ballii (2n=16)+B. juncea (2n=38), were obtained by fusing mesophyll protoplasts of T. ballii and hypocotyl protoplasts of B. juncea using polyethylene glycol. The heterokaryotic fusion frequency was around 23%. Plants were regenerated from 10 out of 2×10⁴ calli of which four were hybrids. Hybrids were intermediate between the parents in general morphology. However, in some characters one of the parents dominated. All the plants were symmetric in their chromosomal constitution as revealed by the formation of 26 bivalents at metaphase-I of meiosis. Two trivalents and 2 univalents were also observed in some pollen mother cells. Hybrid nature was also confirmed by Southern hybridization of DNA of one regenerated plant restricted with Hind III and probed with the nick translated plasmid pTA71 carrying a wheat nuclear r-DNA sequence. Hybrid plant RT 1 showed bands characteristic of both parents. All the plants were absolutely pollen sterile. However, on backcrosses to B. juncea seeds were obtained.     

Synthesis, morphology and cytogenetics of Raphanofortii (TTRR, 2n = 38): a new amphidiploid of hybrid Brassica tournefortii (TT, 2n = 20) ×Raphanus caudatus (RR, 2n=18)

Amphidiploid Raphanofortii was synthesized by colchicinization of the F₁ hybrid Brassica tournefortii (TT, 2n = 20)×Raphanus caudatus (RR, 2n = 18). The crossability between these two species, and the cytomorphology of the F₁ plants and the amphidiploids were investigated. Intergeneric hybrids between the species were obtained only when B. tournefortii was involved as female parent. The hybrid plants were intermediate for most of the morphological attributes and showed very low pollen fertility compared to the parents. Although a majority of the pollen mother cells of the dihaploid hybrid (TR, 2n = 19) harboured univalents, a maximum of six bivalents were also observed. Of the 37 colchicine-treated F₁ plants analyzed cytologically, 21 were found to be true amphidiploids (2n = 38), whereas seven were mixoploids. Meiosis in the amphidiploids was characterized by the occurrence of 19 bivalents, though multivalents and univalents were also observed in a few cells. Most of the amphidiploid plants exhibited a fairly high pollen and seed fertility, which was further enhanced with the advancement of generations. Out of 69 plants investigated in the A₂ generation, 64 were euploids while the remaining five were aneuploids (2n = 36, 37, 39, 40 and 42). The newly synthesized Raphanofortii has great potential as a new commercial crop, as well as a bridge species for the transfer of economically important attributes of both the species to other Brassicas. Received: 2 November 1999 / Accepted: 26 March 2000     

High efficiency production and genomic<Emphasis Type="Italic">in situ</Emphasis> hybridization analysis of<Emphasis Type="Italic">Brassica</Emphasis> aneuploids and homozygous plants

Interspecific and intergeneric hybridizations have been widely used in plant genetics and breeding to construct stocks for genetic analysis and to introduce into crops the desirable traits and genes from their relatives. The intergeneric crosses between Brassica juncea (L.) Czern. & Coss., B. carinata A. Braun and Orychophragmus violaceus (L.) O. E. Schulz were made and the plants produced were subjected to genomic in situ hybridization analysis. The mixoploids from the cross with B. juncea were divided into three groups. The partially fertile mixoploids in the first group (2n = 36-42) mainly contained the somatic cells and pollen mother cells (PMCs) with the 36 chromosomes of B. juncea and additional chromosomes of O. violaceus. The mixoploids (2n = 30-36) in the second and third groups were morphologically quite similar to the mother plants B. juncea and showed nearly normal fertility. The plants in the second group produced the majority of PMCs (2n = 36) with their chromosomes paired and segregated normally, but 1-4 pairs of the O. violaceus chromosomes were included in some PMCs. The plants in the third group produced only PMCs with the 36 B. juncea chromosomes, which were paired and segregated normally. The mixoploids (2n = 29-34) from the cross with B. carinata produced the majority of PMCs (2n = 34) with normal chromosome pairing and segregation, but some plants had some PMCs with 1-3 pairs of chromosomes from O. violaceus and other plants had only PMCs with the B. carinata chromosomes. The Brassica homozygous plants and aneuploids with complete or partial chromosome complements of Brassica parents and various numbers of O. violaceus chromosomes were derived from these progeny plants. The results in this study provided the molecular cytogenetic evidence for the separation of parental genomes which was previously proposed to occur in the hybridizations of these two genera.     

Production and cytogenetics of hybrids of Triticum aestivum x Leymus innovatus

Summary Hybrid plants were obtained between Triticum aestivum (2n=6x=42, AABBDD) and Leymus innovatus (2n=4x=28, JJNN) at a frequency varying from 0.4% to 1.2% of the pollinated florets. Improvement of the embryo culture medium resulted in a higher frequency of embryo rescue. Eight of ten hybrids had the expected chromosome number of 35 (ABDJN). Meiotic analysis indicated that there was no homology between the genomes of the two species. Two hybrids had only 28 chromosomes. Comparison of chromosome pairing between the two types of hybrids suggested that Leymus innovatus carries genes that affect chromosome pairing and behavior. The relatively high occurrence of spontaneous doubling in the meiocytes of these hybrids may indicate that backcrossing of the hybrids to wheat should be possible, although frequent chromosome irregularities observed in the meiocytes of the hybrids may decrease the probability of success of this step, which is essential to the process of gene transfer from L. innovatus to wheat.Contrib. no. 366     

Cellular expression pattern of the glycine decarboxylase P protein in leaves of an intergeneric hybrid between the C3-C4 intermediate species Moricandia nitens and the C3 species Brassica napus

 An intergeneric hybrid plant was produced between the C₃-C₄ intermediate species Moricandia nitens and the C₃ species Brassica napus by sexual hybridization and in vitro embryo rescue. The hybrid nature of the plant was apparent in its morphology and flower pigmentation and was confirmed by leaf isozyme patterns. The overall plant morphology and the shape and thickness of leaves of the hybrid plant were intermediate between those of the parent species. However, the bundle-sheath cells of the hybrid resembled those of the C₃ parent and lacked the organelle development of the C₃-C₄ intermediate parent. Immunogold labelling for the presence of the P subunit of the mitochondrial glycine decarboxylase complex revealed a very similar labelling density on mitochondria in bundle-sheath and mesophyll cells in B. napus, while in  M. nitens the P subunit was only detectable in bundle sheath cells. In the hybrid the labelling density on mesophyll cell mitochondria was almost half of that on the bundle-sheath mitochondria. The CO₂ compensation point of the hybrid was significantly less than that of the C₃ parent but was not as low, nor as responsive to changes in light intensity, as for the C₃-C₄ parent. Received: 23 October 1997 / Accepted: 28 November 1997     

Karyotype stabilization in intergeneric hybrids of the subtribe Triticinae

Summary The C-banded data obtained from Triticinae hybrids are studied with reference to the stabilization of their karyotypes. The types of hybrids distinguished according to genome structure are type I with minimally one diploid genome and type II with a haploid set only. Comparative analysis demonstrates that type I differs from II in karyotype stabilization. The chromosomes from various haploid genomes are combined into new genomes in type I; type II is represented only by amphiploids with the complete set of the chromosomes from all the genomes. The meiotic behaviour of the haploid genome chromosomes were found to have a modifying effect on karyotype stabilization: type II becomes I when homoeologous pairing level is high and when it is associated with the reductional division of univalents.The paper is dedicated to the memory of Professor V. V. Khvostova     

Somatic hybrids between Brassica oleracea L. and Sinapis alba L. with resistance to Alternaria brassicae (Berk.) Sacc.

 Somatic Hybrids between Sinapis alba and rapid-cycling Brassica oleracea were generated for transferring of resistance to Alternaria brassicae to B. oleracea. A. brassicae causes the significant disease black spot in cruciferous crops. A total of 27 plants were regenerated from protoplast fusion using 0, 5, 10, 20 and 30 krad γ-irradiation of the resistance donor and iodoacetate treatment of B. oleracea. All plants showed intermediate morphology with partially divided leaves and some trichomes on stems and leaves. Flow cytometry and banding patterns of the enzymes leucine amino peptidase (LAP) and phosphoglucose isomerase (PGI) confirmed the hybrid status of the regenerated plants. Some of the plants obtained from cuttings from the somatic hybrids showed a resistance to A. brassicae that was similar to that found in S. alba. The flowers of the somatic hybrids had reduced anthers with little pollen production. Received : 9 May 1996 / Accepted : 15 November 1996     

Development and characterisation of Brassica napus-Sinapis arvensis addition lines exhibiting resistance to Leptosphaeria maculans

Blackleg caused by Leptosphaeria maculans is one of the most important diseases affecting oilseed rape worldwide. Sinapis arvensis is valuable for the transfer of blackleg resistance to oilseed rape (Brassica napus) because this species contains high resistance against various aggressive isolates of the blackleg fungus. These include at least one Australian isolate which has been found to overcome resistance originating from species with the Brassica B genome, until now the major source for interspecific transfer of blackleg resistance. Backcross offspring from intergeneric crosses between Brassica napus and S. arvensis were subjected to phytopathological studies and molecular cytogenetic analysis with genomic in situ hybridisation (GISH). The BC₃S progenies included fertile plants exhibiting high seedling (cotyledon) and adult plant resistance associated with the presence of an acrocentric addition chromosome from S. arvensis. In addition, some individuals with adult plant resistance but cotyledon susceptibility were observed to have a normal B. napus karyotype with no visible GISH signals, indicating possible resistant introgression lines. Phytopathological analysis of selfing progenies from 3 different highly resistant BC₃ plants showed that seedling and adult plant resistance are probably conferred by different loci. Received: 20 September 1999 / Accepted: 25 March 2000     

Production and characterization of somatic hybrids between Brassica napus and Raphanus sativus

Intergeneric somatic hybridization between Brassica napus and Raphanus sativus was carried out to enrich gene pool of B. napus. Twelve somatic hybrids were produced via PEG-mediated protoplast fusion between B. napus and R. sativus. The hybridity was confirmed by morphological observation and molecular marker analysis. Hybrid progenies (BC₁) were obtained via backcrosses with B. napus. Behaviour of R. sativus chromosomes in a B. napus background in the F₁ and BC₁ plants was revealed by genomic in situ hybridization (GISH). The potential of somatic hybridization to enrich the suitable gene pool for rapeseed breeding is discussed.     

Comparison of the genetic maps of Brassica napus and Brassica oleracea

 The genus Brassica consists of several hundreds of diploid and amphidiploid species. Most of the diploid species have eight, nine or ten pairs of chromosomes, known respectively as the B, C, and A genomes. Genetic maps were constructed for both B. napus and B. oleracea using mostly RFLP and RAPD markers. For the B. napus linkage map, 274 RFLPs, 66 RAPDs, and two STS loci were arranged in 19 major linkage groups and ten smaller unassigned segments, covering a genetic distance of 2125 cM. A genetic map of B. oleracea was constructed using the same set of RFLP probes and RAPD primers. The B. oleracea map consisted of 270 RFLPs, 31 RAPDs, one STS, three SCARs, one phenotypic and four isozyme marker loci, arranged into nine major linkage groups and four smaller unassigned segments, covering a genetic distance of 1606 cM. Comparison of the B. napus and B. oleracea linkage maps showed that eight out of nine B. oleracea linkage groups were conserved in the B. napus map. There were also regions in the B. oleracea map showing homoeologies with more than one linkage group in the B. napus map. These results provided molecular evidence for B. oleracea, or a closely related 2n=18 Brassica species, as the C-genome progenitor, and also reflected on the homoeology between the A and C genomes in B. napus. Received: 14 June 1996 / Accepted: 11 October 1996     

Characterization of sexual progenies of male-sterile somatic cybrids between Brassica napus and Brassica tournefortii

Cytogenetic studies were performed on four male-sterile progenies derived from four different cybrids produced between Brassica napus and B. tournefortii using the donor-recipient protoplast fusion method. The objective of these studies was to characterize the nuclear constitution of the plants. Mitotic investigation revealed that three of the four male-sterile lines had 38 chromosomes, which is equal to that of B. napus. The fourth line, C6, had variable chromosome numbers, ranging from 39 to 42 in different plants. The meiotic behavior in each progeny varied distinctly. Of the plants having 38 chromosomes, fairly high chromosome pairing, on average 18.08 bivalents per cell, was detected at metaphase-I. However, univalents with an average of 1.39 per cell, and very low frequencies of trivalents and/or tetravalents, were also observed in the lines. These results revealed that male-sterile cybrid lines were obtained with 38 chromosomes and a relatively high level of chromosome-pairing ability, indicating their potential for establishing a stable male-sterile rapeseed line. Received:15 December 1998 / Accepted:30 January 1999     

STS markers linked to Phoma resistance genes of the Brassica B-genome revealed sequence homology between Brassica nigra and Brassica napus

The RFLP and AFLP techniques are laborious and expensive and therefore of limited use for marker-assisted selection, demanding a high throughput of samples in a short time. But marker-assisted selection is most useful for traits which are hard to score on single plants and influenced by environmental factors. Four RFLP and three AFLP markers have been found to be linked to genes of the B-genome of Brassica mediating resistance against Phoma lingam in oilseed rape. One RFLP and one AFLP marker were converted into three PCR-based STS markers: one of dominant, as well as one of codominant inheritance separated in a standard agarose gel and a third one of codominant inheritance to be separated in a polyacrylamide gel on an automated sequencer. As expected, the STS markers mapped at the same position as the original RFLP and AFLP markers. The STS markers are efficient in marker-assisted backcross programs of the resistant B-genome/Brassica napus recombinant lines with most of the tested oilseed rape varieties and breeding lines. More than 90% of the tested oilseed rape varieties and breeding lines exhibited no resistance marker alleles. The mapping results obtained with the markers, as well as comparative sequencing of the marker alleles, indicate synteny and homology between the B-genome resistance gene donors and B. napus in the region of the resistance genes. The location of the resistance genes in the B-genome/B. napus recombinant lines is most likely on the A genome. Thus the transfer of the B-genome resistance genes into Brassica campestris is also possible. Received: 9 December 1999 / Accepted: 21 June 2000