甜叶菊同源四倍体离体诱导及鉴定

用不同浓度秋水仙素溶液处理甜叶菊不定芽,诱导同源四倍体,并进行解剖学、染色体鉴定和流式细胞仪鉴定倍性。结果表明:(1)用0.20%的秋水仙素溶液浸泡甜叶菊不定芽12h,同源四倍体诱导率最高,可达32.14%。(2)同源四倍体植株与二倍体(对照)相比,其气孔、叶片等均表现巨大性,且叶片变厚、叶色浓绿、叶片皱缩。(3)对照植株染色体2n=2x=22,四倍体植株染色体2n=4x=44;流式细胞仪倍性鉴定结果显示,对照DNA相对含量为100,四倍体DNA相对含量为200。(4)该研究共鉴定出48株甜叶菊同源四倍体植株,为进行倍性植株的诱导奠定了技术基础,为进一步开展甜叶菊同源四倍体新品种的选育提供了实验材料。     

Genomic characterisation and the detection of raspberry chromatin in polyploid Rubus

 This paper reports genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) data for chromosomes of raspberry (Rubus idaeus 2n=2x=14), blackberry (Rubus aggregate, subgenus Eubatus. 2n=2–12x=14–84) and their allopolyploid derivatives used in fruit breeding programmes. GISH was used to discriminate labelled chromosomes of raspberry origin from those of blackberry origin in allopolyploid hybrid plants. The raspberry chromosomes were labelled by GISH at their centromeres, and 1 chromosome was also labelled over the short arm. In one allopentaploid plant a chromosome carried a terminal signal. Karyotype analysis indicated that this is a blackberry chromosome carrying a raspberry translocation. GISH analysis of an aneuoctaploid blackberry cv ‘Aurora’ (2n=8x=58) showed that both whole and translocated raspberry chromosomes were present. The basic Rubus genome has one ribosomal DNA (rDNA) locus, and in all but one case all levels of ploidy had the expected multiples of rDNA loci. Interestingly, in the blackberry cv ‘Aurora’, there were only six sites, two less than might be predicted from its aneuoctaploid chromosome number. Our results highlight the potential of GISH and FISH for genomic designation, physical mapping and introgression studies in Rosaceous fruit crops. Received: 20 February 1998 / Accepted: 12 May 1998     

Chromosomal variation in immature embryo derived calluses of barley (Hordeum vulgare L.)

Summary Chromosome counts of ten morphogenic and seven non-morphogenic immature embryo derived calluses of barley,Hordeum vulgare L. cv. Himalaya, were determined. Morphogenic calluses carried the normal chromosome complement (2n=2x=14) in a majority of the cells. A low frequency of haploid (2n=x=7), triploid (2n=3x=21), tetraploid (2n=4x=28) and octoploid (2n=8x=56) cells were also observed. In contrast, non-regenerability of a callus was attributed to the cells having numerical and structural chromosomal changes. In these calluses, aneuploid cells around diploid, triploid, and tetraploid chromosome numbers predominated. It has been demonstrated that chromosomal changes were induced during the culture and that they did not pre-exist in the cultured barley embryos. Based on this study, it is suggested that chromosome analysis of a non-regenerable callus should be conducted before altering the media composition.     

Chromosome size and DNA values in sundews (Droseraceae)

Relative DNA content has been determined Feulgen cytophotometrically and autoradiographically for roottip nuclei of Drosophyllum lusitanicum L., (2n=12), Drosera rotundifolia L. (2x=20), D. intermedia Hayne (2x=20), D. linearis Goldie (2x= 20), D. binata Labill. (3x=32), D. capensis L. (4x= 40), D. spathulata Labill. (8x=80), all Droseraceae. Relative DNA values per diploid genome for Drosophyllum and diploid, triploid, and higher polyploid Drosera were approximately as 16421. These values are terms of a geometric series and are compatible with a multistranded (polyneme) interpretation of chromosome structure.     

Segregation for sexual seed production in Paspalum as directed by male gametes of apomictic triploid plants

BACKGROUND AND AIMS: Gametophytic apomixis is regularly associated with polyploidy. It has been hypothesized that apomixis is not present in diploid plants because of a pleiotropic lethal effect associated with monoploid gametes. Rare apomictic triploid plants for Paspalum notatum and P. simplex, which usually have sexual diploid and apomictic tetraploid races, were acquired. These triploids normally produce male gametes through meiosis with a range of chromosome numbers from monoploid (n = 10) to diploid (n = 20). The patterns of apomixis transmission in Paspalum were investigated in relation to the ploidy levels of gametes. METHODS: Intraspecific crosses were made between sexual diploid, triploid and tetraploid plants as female parents and apomictic triploid plants as male parents. Apomictic progeny were identified by using molecular markers completely linked to apomixis and the analysis of mature embryo sacs. The chromosome number of the male gamete was inferred from chromosome counts of each progeny. KEY RESULTS: The chromosome numbers of the progeny indicated that the chromosome input of male gametes depended on the chromosome number of the female gamete. The apomictic trait was not transmitted through monoploid gametes, at least when the progeny was diploid. Diploid or near-diploid gametes transmitted apomixis at very low rates. CONCLUSIONS: Since male monoploid gametes usually failed to form polyploid progenies, for example triploids after 4x x 3x crosses, it was not possible to determine whether apomixis could segregate in polyploid progenies by means of monoploid gametes.     

Cytotaxonomy and breeding system of the genusBiscutella (Cruciferae)

Chromosome counts were determined for 46 populations ofBiscutella representing 28 taxa. The genus was found to contain diploid taxa with 2n = 12, 16 and 18, tetraploid taxa with 2n = 36 and hexaploid taxa having 2n = 54.B. laevigata L. s. l. consists of diploid and tetraploid populations which are poorly differentiated morphologically. TetraploidB. laevigata s. l. and hexaploidB. variegata Boiss. & Reuter (s. l.) are characterized by chromosomal instability. The variation in chromosome numbers and the occurrence of polyploidy is discussed in relation to the taxonomy of the genus. An investigation of the breeding system showed that most of the annual species were self-compatible and partly inbreeding and most of the perennial species self-incompatible and, therefore, outbreeding, while one annual species,B. cichoriifolia Loisel., showed both systems.     

三种绞股蓝植物染色体观察

本文对分布于陕西省的3种绞股蓝植物进行了染色体观察。其结果：绞股蓝染色体数为2n=2x=22,长梗绞股蓝为2n=2x=22,心籽绞股蓝为2n=6x=66。后两种为本文首次报道。 这些观察结果为我国绞股蓝属植物的分类和推广利用提供了细胞学依据。     

Interspecific hybridization between Vigna radiata (L.) Wilczek and V. glabrescens

Summary Interspecific hybrids of the mungbean, Vigna radiata (L.) Wilczek (2n=22) and V. glabrescens (2n=44) were generated with the aid of embryo culture. V. glabrescens x V. radiata hybrids were recovered via germination of the immature embryos. Reciprocal hybrids were obtained via shoot formation from embryonic callus. The authenticity of the hybrids was determined by morphological characteristics, chromosome number, and isozyme patterns. The hybrids were highly sterile upon selfing, but backcrossing to the diploid parent yielded viable seeds. Some of the plants resembled the diploid parent morphologically while others resembled neither parent. The backcross plants were sufficiently fertile to give a large number of mature, selfed seeds. Plants obtained differed morphologically and in their isozyme patterns from either parent, indicating introgression. These progeny populations will be used as bridging materials to transfer pest resistance from the wild tetraploid to the cultivated mungbean.     

Systematics and karyoevolution inMagnoliidae:Tetrameranthus as compared with otherAnnonaceae genera of the same chromosome number

First generic chromosome counts reveal the base number x=7 for the generaTetrameranthus andRollinia. T. umbellatus from the Peruvian Amazon is diploid (2n=14),T. duckei from Brazil (Manaus) is tetraploid (2n=28). In the NeotropicsRollinia (7 species counted) has developed diploid to octoploid taxa (2n=14, 28, 42, 56). Counts of 7 South AmericanAnnona species are presented for comparison (2n=14, 28). The West AfricanCleistopholis patens has 2n=14. The Asian genusMezettia: 2n=14 and the neotropicalGuatteria tribe: 2n=28 are also revised. A detailed karyomorphological comparison, including karyotypes, banding patterns, condensing behaviour of chromosomes and structure of interphase nuclei reveals that the closely related generaAnnona andRollinia are almost identical in their diploid genomes, whereas the polyploid ones differ in their heterochromatin (=hc) composition and number of NO-chromosomes.Cleistopholis, Mezettia and theGuatteria tribe are karyologically and systematically distinct from each other and fromAnnona/Rollinia. Tetrameranthus as compared with the karyomorphology of about 60 other Annonaceous genera has a very peculiar and unusual karyomorphology which underlines its isolated position. Nuclear structures are almost identical in the African genusUvariopsis (2n = 16) and partly similar in theGuatteria tribe; both also share some morphological similarities and possibly are related. From a comparison ofTetrameranthus with several nuclear types within theMagnoliidae, a new model of chromosome evolution in primitive Angiosperms is suggested. In respect to their eco-morphological differentiation the genera investigated differ strongly from each other.Dedicated to Prof. Dr. K.-H.Rechinger on the occasion of His 80th birthday.     

三种绞股蓝植物染色体观察

本文对分布于陕西省的3种绞股蓝植物进行了染色体观察,其结果:绞股蓝染色体数为2n=2x=22,长梗绞股蓝为2n=2x=22,心籽绞股蓝为2n=6x=66。后两种为本文首次报道,这些观察结果为我国绞股蓝属植物的分类和推广利用提供了细胞学依据。     

Duplication of the chromosome number of diploid Brachiaria brizantha plants using colchicine

 Some species of Brachiaria, generally tetraploid apomictic varieties, have become important forage grasses in the tropics. Breeding of Brachiaria depends on compatibility with the available apomitic tretraploid cultivars. This paper describes a procedure for chromosome duplication of two Bracharia brizantha diploid sexual accessions, using colchicine treatment of basal segments of in-vitro-grown plants. Explants were cultured on a medium containing 1 mg/l naphthaleneacetic acid, 3 mg/l kinetin and 0.01% colchicine for 48 h and transferred to the same medium without colchicine until shoot regeneration occurred. Regenerated plants were screened by flow cytometry, and chromosome number duplication was confirmed by cytological analysis of root tips. Received: 9 Ocotber 1997 / Revision received: 9 December 1998 / Accepted: 15 June 1999     

Diploidization in megagametophyte-derived cultures of the gymnosperm Larix decidua

Tested haploid embryogenic lines (n=12) of Larix dedicua Mill, initiated from megagametophyte tissue were maintained on half-strength LM medium without growth regulators. The cultures were analyzed for ploidy level after 1–9 years. All lines tested were found to have doubled (2n=24) their chromosome number at the end of the experiment, though there were a few lines that still gave occasional haploid counts. Flow cytometric data of embryogenic tissue confirmed these results. Protoplasts were stained in ethidium bromide, and cultured human leucocytes and chicken erythrocytes were used as internal standards. Haploid megagametophytes from immature seeds of L. decidua and known diploid culture lines of a related hybrid (L. x eurolepis) were also analyzed by flow cytometry. Haploid reference material had 12.3–13.6 pg DNA per cell, whereas formerly haploid callus lines had an average of 25.0 pg DNA per cell. The one exception was a known, genetically unstable line of L. decidua (34.8 pg DNA per cell). The diploid cell line of L. x eurolepis had 27.6 pg DNA per cell. The results show that spontaneous diploidization of megagametophyte lines is relatively rapid and that both haploid and dihaploid lines are embryogenic in larch.     

The nature of the genetic control of Endosperm Balance Number based on aneuploid analysis of Datura

 The genetic control of Endosperm Balance Number (EBN), a mechanism of effective ploidy that controls seed development, was studied using aneuploidy. The Endosperm Balance Number hypothesis proposes that each species has an effective ploidy (EBN) in the endosperm and that it is the effective ploidies, rather than the numerical (actual) ploidies, that must be in a 2:1 maternal to paternal ratio for normal endosperm development. Experiments were conducted in Datura stramonium L. (2n=4x=48) to determine if more than one chromosome but less than the whole genome could change the EBN of the female. Triploids were crossed with tetraploids to produce aneuploids. Most plump seeds gave rise to 2n=4x=48 chromosome plants. Six plants had between 38 and 47 chromosomes. Karyotyping of these plants supported the conclusion that only two chromosomes (1.2 and 19.20), when extra, were necessary to change the EBN of the central cell. Received: 25 April 1998 / Revision accepted: 25 January 1999     

In situ hybridization identifies the diploid progenitor species of Coffea arabica (Rubiaceae)

 The most important commercial coffee species, Coffea arabica, which is cultivated in about 70% of the plantations world-wide, is the only tetraploid (2n=4x=44) species known in the genus. Genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) were used to study the genome organization and evolution of this species. Labelled total genomic DNA from diploid species (C. eugenioides, C. congensis, C. canephora, C. liberica) closely related to C. arabica was separately used as a probe in combination with or without blocking DNA to the chromosome spreads of C. arabica. GISH discriminated between chromosomes of C. arabica only in the presence of an excess of unlabelled block DNA from the species not used as a probe. Among the range of different species combinations used, DNA from C. eugenioides strongly and preferentially labelled 22 chromosomes of the tetraploid C. arabica, while the remaining 22 chromosomes were labelled with C. congensis DNA. The similarity of observations between C. arabica and the two diploid species using two ribosomal genes with FISH with respect to metaphase chromosomes provided additional support to the GISH results. These results confirm the allopolyploid nature of C. arabica and show that C. congensis and C. eugenioides are the diploid progenitors of C. arabica. Received: 2 February 1998 / Accepted: 12 May 1998     

铁皮石斛四倍体离体诱导和鉴定及生理特性研究

以铁皮石斛原球茎为材料,经不同质量浓度的秋水仙素(C₂₂H₂₅O₆)和0.02 g·mL^-1二甲基亚砜(DMSO)混合水溶液处理后进行组织培养,通过对变异株进行形态学、细胞学及流式细胞仪鉴定,以期获得稳定的四倍体植株并分析其生理特性。结果表明:用2.0 g·L^-1秋水仙素和0.02 g·mL^-1 DMSO混合水溶液处理铁皮石斛原球茎36 h后,植株诱导率达20%;诱导四倍体植株在形态上明显矮化、茎秆粗壮、叶片变小增厚、气孔直径增大;细胞遗传学观察发现,四倍体植株染色体2n=4x=76,二倍体植株染色体2n=2x=38;流式细胞仪分析显示,DNA相对含量四倍体为400,二倍体仅为200;四倍体植株叶片中叶绿素含量、可溶性蛋白、可溶性糖含量均高于二倍体,分别为5.03、3.59、2.98 mg·g^-1;四倍体叶片中主要抗氧化酶POD和SOD活性均显著高于二倍体,分别为9.08、180.4 U·mg^-1,且四倍体植株明显降低了MDA含量累积。研究认为,2.0 g·L^-1秋水仙素和0.02 g·mL^-1 DMSO混合水溶液处理原球茎36 h可提高诱导成功率、降低嵌合体比例,此组合为诱导四倍体较佳诱导条件。     

Rapid regeneration of whole plants in large crabgrass (Digitaria sanguinalis L.) using thin-cell-layer culture

A method was designed to produce rapidly (10–14 days) and directly (without intermediate callus) whole plants of Digitaria sanguinalis with a high yield without subculture. These plants developed from new structures, designated “pseudo-embryogenic structures”, initiated only 1 week after the culture of tranverse thin cell layers (tTCLs), i.e., thin stem sections, on a Murashige and Skoog medium containing 3% sucrose and a combination of a low concentration of 2,4-dichlorophenoxyacetic acid (1 μm) and a high concentration of 6-benzylaminopurine (10 μm). The fresh weight of plants regenerated per tTCL on gelrite was 6 times higher than with agar and 30 times higher than with agarose. Received: 25 August 1997 / Revision received: 18 February 1998 / Accepted: 10 March 1998     

Post-zygotic gametic selection due to endosperm balance number explains unusual chromosome numbers of 3×× 2× progeny in Solanum

 Crosses between triploid and diploid genotypes are usually the best sources of trisomics in potato as well as in several other crop species. However, 3×× 2× crosses between triploid (2n=3×=36; 2EBN) Solanum commersonii-S. tuberosum hybrids and diploid (2n= 2×=24; 2EBN) genotypes gave progenies with a high number of extra chromosomes, 29–36, suggesting that only eggs with 17–24 chromosomes produced embryos that reached full development. Our hypothesis is that although triploids produce eggs with a range of chromosome numbers, 3×× 2× crosses involving a 2×(2EBN) parent favor eggs with a high chromosome number. These eggs have higher probabilities of possessing the same endosperm balance number (EBN) value (i.e. 1) of gametes produced by the 2EBN diploid parent to give the required 2:1 maternal to paternal EBN ratio in the hybrid endosperm. Under this model, trisomics are produced only if the diploid parent has an EBN of 1. Based on our results and those reported in the literature, it is proposed that in 3×(2EBN) × 2×(2EBN) crosses the endosperm balance number exercises negative selection for gametes with a low chromosome number, and a corresponding low EBN, and positive selection for gametes with a high chromosome number and EBN. Received: 2 April 1998 / Revision accepted: 27 October 1998     

Decaploidy in Fragaria iturupensis (Rosaceae)

The strawberry genus, Fragaria (Rosaceae), has a base chromosome number of x = 7. Cultivated strawberries (F. ×ananassa nothosubsp. ananassa) are octoploid (2n = 8x = 56) and first hybridized from F. chiloensis subsp. chiloensis forma chiloensis × F. virginiana subsp. virginiana. Europe has no known native octoploid species, and only one Asian octoploid species has been reported: F. iturupensis, from Iturup Island. Our objective was to examine the chromosomes of F. iturupensis. Ploidy levels of wild strawberry species, include diploid (2n = 2x = 14), tetraploid (2n = 4x = 28), pentaploid (2n = 5x = 35), hexaploid (2n = 6x = 42), octoploid (2n = 8x = 56), and nonaploid (2n = 9x = 63). Artificial triploid (2n = 3x = 21), tetraploid, pentaploid, octoploid, decaploid (2n = 10x = 70), 16-ploid, and 32-ploid plants have been constructed and cultivated. Surprisingly, chromosome counts and flow cytometry revealed that F. iturupensis includes natural decaploid genotypes with 2n = 10x = 70 chromosomes. This report is the first of a naturally occurring decaploid strawberry species. Further research on F. iturupensis and exploration on northern Pacific islands is warranted to ascertain the phylogeny and development of American octoploid species.     

Somatic embryogenesis and plant regeneration in diploid and triploid Arachis pintoi

Plants of two cytotypes (2n=2x=20, and 2n=3x=30) of pinto peanut (Arachis pintoi Krapov. & W.C. Gregory) were regenerated through somatic embryogenesis. Embryogenic calli were induced from shoot tips or immature leaves dissected from in vitro growing plants. In the case of the diploid peanut the best somatic embryogenesis was achieved when shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 10 mg dm⁻³ Picloram (PIC) and 0.1 mg dm⁻³ 6-benzylaminopurine (BAP) or when explants from immature leaves were cultured on MS + 10 mg dm⁻³ PIC + 0.01 mg dm⁻³ BAP. In the case of triploid peanut the highest number of somatic embryos was obtained when shoot tips were cultured on MS + 10 mg dm⁻³ PIC + 0.01 mg dm⁻³ BAP or when immature leaves were cultured on MS + 20 mg dm⁻³ PIC + 0.01 mg dm⁻³ BAP. Somatic embryos were converted into plants by culture on MS + 0.01 mg dm⁻³ naphthaleneacetic acid + 0.01 mg dm⁻³ BAP. Plants were successfully transferred to pots in greenhouse.