Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

Karyological relationships among some South American species of Solanum (Solanaceae) based on fluorochrome banding and nuclear DNA amount

Fluorescent chromosome banding and measurements of nuclear DNA content by image cytometry of Feulgen-stained cells were performed in one sample each of eight diploid (2n?=?24) species of Solanum: S.?endoadenium, S.?argentinum, S.?pseudocapsicum, S.?atropurpureum, S.?elaeagnifolium, S.?sisymbriifolium, S.?chenopodioides, and S.?palustre. The species studied could be distinguished by heterochromatin amount, banding patterns, and genome size. They exhibited only GC-rich heterochromatin and showed a comparatively low heterochromatin amount (expressed as percentage of haplotype karyotype length), ranging from 2.10 in S.?argentinum to 8.37 in S.?chenopodioides. Genome size displayed significant variation between species, with 1C-values ranging from 0.75?pg (735?Mbp) in S.?palustre to 1.79?pg (1,754?Mbp) in S.?sisymbriifolium. No significant correlation between genome size and heterochromatin amount was observed, but intrachromosomal asymmetry index (A ₁) was negative and significantly correlated with heterochromatin amount. DNA content was positively and significantly correlated with karyotype length. DNA C-value distribution in the genus as well as karyotype affinities and relationships between species are discussed in relation to different infrageneric classifications of Solanum.     

Genome size and environmental factors in the genus Pinus

Nuclear 1C DNA content in haploid megagametophyte tissue of 18 North American and one exotic Pinus species was determined using scanning microspectrophotometry. The nuclear DNA content in root meristematic cells of Zea mays L. ssp. mays, inbred line Va35 (4C = 10.31 pg) was used as a standard. DNA content measured by microspectrophotometry was verified using laser flow cytometry with two additional standards, Hordeum vulgare cv. Sultan (2C = 11.12 pg) and P. eldarica (2C = 47.30 pg). DNA values obtained by both methods were significantly correlated (r = 0.987). The 1C nuclear DNA content ranged from 21 pg to 31 pg. The ratio of DNA content in embryo tissue of P. eldarica to that in megagametophyte tissue was 1.72 by scanning microspectrophotometry and 1.74 by laser flow cytometry. To date, this is the most comprehensive data set available for North American Pinus species. Relationships between genome size of 18 North American Pinus species and climatic factors and indices of growth were investigated using regression and correlation analyses. Positive correlations were observed between nuclear DNA content and growth indices, minimum seed-bearing age, and seed dimensions. Strong negative correlations were observed between nuclear DNA content and two climatic factors, the lowest mean annual and monthly precipitation (excluding January) and the highest mean monthly spring air temperature. These correlations suggest that the large genome size and its variation in Pinus are adapted responses to the habitats of these species.     

Quantitative DNA variation between and within chromosome complements of Vigna species (Fabaceae)

Cytogenetical investigations, so far, on the organisation and evolution of the genomes of Vigna species have proved difficult due to small chromosome size, large chromosome number and uniformity in chromosome shape and size within and between the complements. In this investigation the nature and extent of DNA variation between thirteen diploid and one polyploid species have been estimated. The DNA variation between diploid species was small and species clustered around a mean value of 2.7 pg. The polyploid species had a greater DNA value of 4.95 pg. No significant variation in 2C DNA content was found between accessions of V. radiata. A comparison of the distribution of DNA among the chromosomes within complements has shown that the excess DNA acquired in evolution was distributed evenly in all chromosomes despite significant differences in chromosome size. The relative changes in chromatin area and DNA density which accompany evolutionary DNA variation was also compared.     

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.     

Change in nuclear DNA content and pollen size with polyploidisation in the sweet potato (Ipomoea batatas,Convolvulaceae) complex

• Genome size evolution and its relationship with pollen grain size has been investigated in sweet potato (Ipomoea batatas), an economically important crop which is closely related to diploid and tetraploid species, assessing the nuclear DNA content of 22 accessions from five Ipomoea species, ten sweet potato varieties and two outgroup taxa.
• Nuclear DNA amounts were determined using flow cytometry. Pollen grains were studied using scanning and transmission electron microscopy.
• 2C DNA content of hexaploid I. batatas ranged between 3.12–3.29 pg; the mean monoploid genome size being 0.539 pg (527 Mbp), similar to the related diploid accessions. In tetraploid species I. trifida and I. tabascana, 2C DNA content was, respectively, 2.07 and 2.03 pg. In the diploid species closely related to sweet potato e.g. I. ×leucantha, I. tiliacea, I. trifida and I. triloba, 2C DNA content was 1.01–1.12 pg. However, two diploid outgroup species, I. setosa and I. purpurea, were clearly different from the other diploid species, with 2C of 1.47–1.49 pg; they also have larger chromosomes. The I. batatas genome presents 60.0% AT bases.
• DNA content and ploidy level were positively correlated within this complex. In I. batatas and the more closely related species I. trifida, the genome size and ploidy levels were correlated with pollen size. Our results allow us to propose alternative or complementary hypotheses to that currently proposed for the formation of hexaploid Ipomoea batatas.

     

Variation in chromosomal DNA associated with the evolution of Arachis species.

The 2C nuclear DNA amounts were determined for 99 accessions, representing 23 Arachis species from 8 of 9 taxonomic sections, and two synthetic amphidiploids. Mean 2C DNA amounts varied by 15.20%, ranging from 10.26 to 11.82 pg, between accessions of Arachis hypogaea (2n = 4x = 40). Nuclear DNA content variation (5.33-5.91 pg) was also detected among Arachis duranensis (2n = 2x = 20) accessions. The intraspecific variation in the two species may have resulted from indirect selection for favourable genome sizes in particular environmental conditions. The accessions belonging to A. hypogaea ssp. hypogaea (mean value 11.27 pg) with longer life cycle had significantly larger mean DNA content than the accessions of A. hypogaea ssp. fastigiata (mean value 10.97 pg). For 20 diploid (2n = 2x = 20) species of the genus, 2C nuclear DNA amounts ranged from approximately 3 to 7 pg. The diploid perennial species of section Arachis have about 12% more DNA than the annual species. Comparisons of DNA amounts show that evolutionary rating is not a reliable guide to DNA amounts in generic sections of the genus; lower DNA values with evolutionary advancement were found in sections Heteranthae and Triseminatae, but the same was not true for sections Arachis and Caulorrhizae. Similarly, there is evidence of significant differences in DNA content between 4 ancient sections (Procumbentes, Erectoides, Rhizomatosae, and Extranervosae) of the genus. The occurrence of genome size plasticity in both A. duranensis and A. hypogaea provides evidence that A. duranensis could be one of the diploid progenitors of A. hypogaea. The DNA content in the two synthetic amphidiploids corresponded to the sum value estimated for parental species. Key words : Arachis species, genome size, Arachis hypogaea, Arachis duranensis, intraspecific variation.     

Is genome downsizing associated with diversification in polyploid lineages of Veronica?

The study of genome size evolution in a phylogenetic context in related polyploid and diploid lineages can help us to understand the advantages and disadvantages of genome size changes and their effect on diversification. Here, we contribute 199 new DNA sequences and a nearly threefold increase in genome size estimates in polyploid and diploid Veronica (Plantaginaceae) (to 128 species, c. 30% of the genus) to provide a comprehensive baseline to explore the effect of genome size changes. We reconstructed internal transcribed spacer (ITS) and trnL‐trnL‐trnF phylogenetic trees and performed phylogenetic generalized least squares (PGLS), ancestral character state reconstruction, molecular dating and diversification analyses. Veronica 1C‐values range from 0.26 to 3.19 pg. Life history is significantly correlated with 1C‐value, whereas ploidy and chromosome number are strongly correlated with both 1C‐ and 1Cx‐values. The estimated ancestral Veronica 1Cx‐value is 0.65 pg, with significant genome downsizing in the polyploid Southern Hemisphere subgenus Pseudoveronica and two Northern Hemisphere subgenera, and significant genome upsizing in two diploid subgenera. These genomic downsizing events are accompanied by increased diversification rates, but a ‘core shift’ was only detected in the rate of subgenus Pseudoveronica. Polyploidy is important in the evolution of the genus, and a link between genome downsizing and polyploid diversification and species radiations is hypothesized. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 243–266.     

Condensed chromatin in diploid and allopolyploid microseris species with different genome size: a quantitative electron microscopic study

Summary The species-specific proportion of chromatin in the condensed state was estimated by quantitative electron microscopic morphometry of nuclear sections in 9 diploid and 5 allopolyploid species of Microseris (Asteraceae). A positive correlation between the genome size (haploid DNA content, or C value) and the percentage of chromatin in the condensed state (as visible in ultrathin sections) was found in diploids (r=0.89). Nuclei of allopolyploid (tetraploid) species exhibit condensed chromatin in a percentage which corresponds to the average of the values found in the parents. This suggests that each parental genome controls chromatin condensation at interphase independently within the nucleus, and that the degree of condensation is not directly determined by the nuclear DNA content per se. Genome size differences among Microseris species may depend preferentially, but not entirely, on DNA fractions located in, and perhaps being the cause of, condensed chromatin.Dedicated to Professor F. Mechelke in honour of his 60^th birthday.     

Spatial and temporal expression of the two sucrose synthase genes in maize: immunohistological evidence

Summary The DNAs of two diploid species of Gossypium, G. herbaceum var. africanum (A₁ genome) and G. raimondii (D₅ genome), and the allotetraploid species, G. hirsutum (A_h and D_h genomes), were characterized by kinetic analyses of single copy and repetitive sequences. Estimated haploid genome sizes of A₁ and D₅ were 1.04 pg and 0.68 pg, respectively, in approximate agreement with cytological observations that A genome chromosomes are about twice the size of D genome chromosomes. This differences in genome size was accounted for entirely by differences in the major repetitive fraction (0.56 pg versus 0.20 pg), as single copy fractions of the two genomes were essentially identical (0.41 pg for A₁ and 0.43 pg for D₅). Kinetic analyses and thermal denaturation measurements of single copy duplexes from reciprocal intergenomic hybridizations showed considerable sequence similarity between A₁ and D₅ genomes (77% duplex formation with an average thermal depression of 6 °C). Moreover, little sequence divergence was detectable between diploid single copy sequences and their corresponding genomes in the allotetraploid, consistent with previous chromosome pairing observations in interspecific F₁ hybrids.Journal paper No. 4461 of the Arizona Agricultural Experiment Station     

Flow cytometry confirms reticulate evolution and reveals triploidy in Central European Diphasiastrum taxa (Lycopodiaceae, Lycophyta)

Background and Aims

Interspecific Diphasiastrum hybrids have been assumed to be homoploid and to produce well-formed spores serving sexual reproduction. If this were the case, forms intermediate between hybrids and parents or hybrid swarms should be expected. The purpose of this study was: (1) to check whether homoploidy consistently applies to the three hybrids throughout their Central European range; (2) to examine whether their genome sizes confirm their parentage as assumed by morphology; and (3) to perform a screening for detection of ploidy levels other than diploid and variation in DNA content due to backcrossing.

Methods

Flow cytometry was used first to measure the relative DNA values [with 4′,6-diamidino-2-phenylindole (DAPI) staining] and ploidy level as a general screening, and secondly to determine the absolute DNA 2C values [with propidium iodide (PI) staining] in a number of selected samples with the main focus on the hybrids.

Key Results

A considerable variation of DNA 2C values (5·26–7·52 pg) was detected between the three European Diphasiastrum species. The values of the diploid hybrids are highly constant without significant variation between regions. They are also intermediate between their assumed parents and agree closely with those calculated from their putative parents. This confirms their hybrid origin, assumed parentage and homoploid status. Considerably higher DNA amounts (9·48–10·30 pg) were obtained for three populations, suggesting that these represent triploid hybrids, an interpretation that is strongly supported by their morphology.

Conclusions

Diploid hybrids have retained their genetic and morphological identites throughout their Central European range, and thus no indications for diploid backcrossing were found. The triploid hybrids have probably originated from backcrossing between a diploid gametophyte of a hybrid (derived from a diplospore) and a haploid gametophyte of a diploid parental species. By repeated crossing events, reticulate evolution patterns arise that are similar to those known for a number of ferns.     

The determination of genome size in male and female germ cells of Drosophila melanogaster by DNA-feulgen cytophotometry

Summary The amounts of DNA in haploid and diploid cells of Drosophila melanogaster have been determined by DNA-Feulgen cytophotometry, using Xenopus laevis erythrocyte nuclei as a reference standard. The haploid male genome is estimated to be 0.18 pg DNA and the haploid female genome, 0.20 pg DNA.     

Evidence for dosage compensation in parthenogenetic Hymenoptera

Amount of DNA-Feulgen staining in individual somatic nuclei and mature sperm of the parthenogenetic wasps, Habrobracon juglandis, H. serinopae, and Mormoniella vitripennis, were determined with a scanning microdensitometer. The haploid genome for both species of Habrobracon was estimated to be 0.15–0.16×10^–12 g DNA, corresponding to a molecular weight of roughly 10×10¹⁰ daltons. The haploid genome of M. vitripennis is approximately twice this value, 0.33–0.34×10^–12 g, or about 20×10¹⁰ daltons. Measurements made on dividing nuclei from syncytial preblastoderm embryos of H. juglandis and M. vitripennis showed that the chromosomes of impaternate males were present in the haploid number and contained the C amount of DNA; whereas nuclei from female preblastoderm embryos contained the diploid number of chromosomes and the 2C amount of DNA. However, hemocyte and brain cell nuclei from either male or female adult wasps contained 2C and 4C amounts of DNA. Both sexes also showed equivalent levels of polyploidy (8C, 16C, or 32C) in Malpighian tubule nuclei. Therefore, in these parthenogenetic species, a mechanism must exist that compensates during later development for the initial two-fold difference in the chromatin content of somatic nuclei in haploid male and diploid female embryos. Hemocytes from impaternate Mormoniella diploid males and triploid females contain the 2C and 3C amounts of DNA, respectively. Therefore dosage compensation involves an additional cycle of DNA replication only in haploid cells, and it insures that a certain minimum quantity of DNA is received by each somatic cell.     

Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

Nuclei were isolated from leaf tissue of differentCapsicum species and the relative fluorescence intensity was measured by flow cytometry after propidium iodide staining.Pisum sativum nuclei with known nuclear genome size (9.07 pg) were used as internal standard to determine nuclear DNA content of the samples in absolute units. The 2C DNA contents ranged between 7.65 pg inC. annuum and 9.72 pg inC. pubescens, and the general mean of the genus was 8.42 pg. These values correspond, respectively, to 1C genome size of 3.691 (C. annuum), 4.690 (C. pubescens) and 4.063 (general mean) Mbp. In general, white-flowered species proved to have less DNA, with the exception ofC. praetermissum, which displayed a 2C DNA content of 9.23 pg. It was possible to divide the studied species into three main groups according to their DNA content, and demonstrate differences in DNA content within two of the three species complexes established on the basis of morphological traits.     

PLOIDY ANALYSIS OF THE TWO MOTILE FORMS OF CHRYSOCHROMULINA POLYLEPIS (PRYMNESIOPHYCEAE)1

In some cultures of the flagellate Chrysochromulina polylepis Manton et Parke, established from cells isolated from the massive bloom in Skagerrak and Kattegat in 1988, we observed, two motile cell types. They were termed authentic and alternate cells and differed with respect to scale morphology. To investigate whether or not the two cell forms were joined in a sexual life cycle, the relative DNA content per cell and relative size of cells of several clonal cultures of C. polylepis were determined by flow cytometry. Percentages of authentic and alternate cells in the cultures were estimated by transmission electron microscopy. Pure authentic cultures (α) contained cells with the lowest level of DNA and were termed haploid. Two pure alternate cultures (β) contained cells with double the DNA content of authentic cells and were termed diploid. Other pure alternate cultures contained haploid cells only, or both haploid and diploid cells. Three cell types were observed, each capable of vegetative propagation: authentic haploid, alternate haploid, and alternate diploid cells. Both the haploid and diploid alternate cells were larger than the haploid authentic cells. Cultures containing diploid cells appeared unstable: cell type ratio and ploidy ratio changed during the experiment where this cell type was present, particularly when grown in continuous light. In contrast, cultures with only haploid cells remained unchanged at all growth conditions tested. Light condition may influence cell type ratio and ploidy ratio. Our attempt to induce syngamy by mixing different authentic haploid clones did not result in mating. Assuming that the authentic and alternate cell types are of the same species, the life cycle of C. polylepis includes three flagellated scale-covered cell forms. Two of the cell types are haploid and may function as gametes, and the third is diploid, possibly being the result of syngamy.     

Intraspecific genome size variation and morphological differentiation of Ranunculus parnassifolius (Ranunculaceae), an Alpine–Pyrenean–Cantabrian polyploid group

The aim of this study was to assess genome size variation and multivariate morphometric analyses to ascertain cytotype distribution patterns and the morphological differentiation within the Ranunculus parnassifolius group in the Pyrenees and the Alps. Although divergences in nuclear DNA content among different species within a genus are widely acknowledged, intraspecific variation is still a somewhat controversial issue. Holoploid and monoploid genome sizes (C‐ and Cx‐values) were determined using propidium iodide flow cytometry in 125 plants of R. parnassifolius s.l. distributed across four European countries. Three different DNA ploidy levels were revealed in the study area: diploid (2n ～ 2x, 57.14%), triploid (2n ～ 3x, 1.19%), and tetraploid (2n ～ 4x, 41.67%). The mean population 2C‐values ranged from 8.15 pg in diploids to 14.80 pg in tetraploids, representing a ratio of 1 : 1.8. Marked intraspecific/interpopulation differences in nuclear DNA content were found. Diploid populations prevail in the Pyrenees, although tetraploid cytotypes were reported throughout the distribution area. In general, mixed‐cytotype populations were not found. The Spearman correlation coefficient did not reveal significant correlations between genome size and altitude, longitude, or latitude. Morphometric analyses and cluster analyses based on genome size variation revealed the presence of three major groups, which exhibited a particular biogeographical pattern. A new cytotype, DNA triploid, was found for the first time. Tetraploid populations showed constant nuclear DNA levels, whereas diploid populations from the Pyrenees, in which introgressive hybridization is suggested as a presumable trigger for genome size variation, did not. Scenarios for the evolution of geographical parthenogenesis in R. parnassifolius s.l. are discussed. Finally, the different levels of effectiveness between plant and animal reference standards are analysed. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 251–271.     

Genome size and genome evolution in diploid Triticeae species.

One of the intriguing issues concerning the dynamics of plant genomes is the occurrence of intraspecific variation in nuclear DNA amount. The aim of this work was to assess the ranges of intraspecific, interspecific, and intergeneric variation in nuclear DNA content of diploid species of the tribe Triticeae (Poaceae) and to examine the relation between life form or habitat and genome size. Altogether, 438 plants representing 272 lines that belong to 22 species were analyzed. Nuclear DNA content was estimated by flow cytometry. Very small intraspecific variation in DNA amount was found between lines of Triticeae diploid species collected from different habitats or between different morphs. In contrast to the constancy in nuclear DNA amount at the intraspecific level, there are significant differences in genome size between the various diploid species. Within the genus Aegilops, the 1C DNA amount ranged from 4.84 pg in A. caudata to 7.52 pg in A. sharonensis; among genera, the 1C DNA amount ranged from 4.18 pg in Heteranthelium piliferum to 9.45 pg in Secale montanum. No evidence was found for a smaller genome size in annual, self-pollinating species relative to perennial, cross-pollinating ones. Diploids that grow in the southern part of the group's distribution have larger genomes than those growing in other parts of the distribution. The contrast between the low variation at the intraspecific level and the high variation at the interspecific one suggests that changes in genome size originated in close temporal proximity to the speciation event, i.e., before, during, or immediately after it. The possible effects of sudden changes in genome size on speciation processes are discussed.     

Karyotype analysis reveals interspecific differentiation in the genus Cedrus despite genome size and base composition constancy

The DNA content and GC% of the four true cedar (Cedrus) species, C. atlantica, C. brevifolia, C. deodara and C. libani, were assessed. Genome size was homogeneous among representative populations of the four species with an average of 32.6±0.6 pg per 2 C or 15.7×10⁹ base pairs per 1 C. The composition in GC was calculated to be 40.7%. A simple monosomatic haploid level was found in the megagametophyte, as compared to the diploid level of the corresponding embryo. Cytogenetic studies showed a diploid chromosome number of 2n=2x=24 in 11 populations sampled over the four species. The chromosome complements have similar morphology and symmetry. However, fluorochromes revealed specific banding patterns in each of the four cedar species. Eight GC-rich chromomycin A₃ bands were observed in Cedrus deodara chromosomes, six in both Cedrus libani and Cedrus brevifolia, and four bands in Cedrus atlantica chromosomes. Moreover, Hoechst 33258 fluorochrome revealed AT-rich sequences specifically located in the centromeric regions while the GC-rich sequences appeared negatively stained. These investigations provide a systematic characterisation of the Cedrus genus and should contribute towards clarification of the phylogenetic relationships among the four species. Received: 10 October 2000 / Accepted: 20 March 2001     

Cytometric determination of genome size and base composition of tree species of three genera of Casuarinaceae

The genome size and base composition of diploid plant species from three genera of the Casuarinaceae family were determined by flow cytometry. Casuarina glauca Sieb. ex Spring. and Gymnostoma deplancheana (Miq.) L. Johnson showed a small genome with 2C = 0.70 pg, 58.6% AT, 40.5% GC for the first species and 2C = 0.75 pg, 58.7% AT, 40.5% GC for the second. Allocasuarina verticillata (Lam.) L. Johnson had a larger genome: 2C = 1.90 pg, 59.3% AT, 41.1% GC. One haploid genome of C. glauca is therefore about 340×10⁶ base pairs. In leaves, roots or bark of these three species, polysomaty was virtually absent: a maximum frequency of 4C nuclei of only 0.08 was found in bark of C. glauca. The genome sizes of C. glauca and G. deplancheana are among the smallest described for higher plants. Small genome size, diploidy and the absence of polysomaty are advantageous traits for facilitating molecular approaches to improvement of these actinorhizal plants and developing the study of their symbiotic interactions with Frankia. Received: 20 December 1997 / Revision received: 13 March 1998 / Accepted: 30 March 1998     

Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics

In the Leguminosae plant family, few of the individual plant species have been used for plant molecular biology research. Among the species investigated no obvious representative ‘model’ legume has emerged. Here a member of the tribe Loteae, Lotus japonicus (Regel) Larsen is proposed as a candidate. L. japonicus is a diploid, autogamous species, with a good seed set, and a generation time of approximately 3 months. The haploid genome consists of six chromosomes and the genome size was estimated to be relatively small (0.5 pg per haploid complement). L. japonicus is susceptible to Agrobacterium tumefaciens and transgenic plants can be regenerated after hygromycin or kanamycin selection. Tissue culture conditions and procedures for transformation and regeneration are described. Stable transformation is demonstrated by segregation of the hygromycin selectable marker after selfing of transgenic plants or test crosses. The possibility of mapping polymorphic DNA markers inbred lines of L. japonicus is also discussed.