Production and genetic characterization of somatic hybrids between leaf mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) and broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

Inter-specific somatic hybrids of leaf mustard (Brassica juncea) and broccoli (Brassica oleracea) were established to introduce cytoplasmic male sterility (CMS) and Verticillium dahliae Kleb. resistance from broccoli to leaf mustard. Protoplasts isolated from hypocotyls and cotyledons of inbred lines of leaf mustard and broccoli were fused using 40% (w/v) polyethelene glycol and then cultured in modified k8p medium supplemented with 0.2 mg/l 2,4-dichlorophenoxi-acetic acid, 0.5 mg/l 6-benzyladenine (BA), 0.1 mg/l naphthaleneacetic acid (NAA), and 0.1 mg/l kinetin (Kin), and 0.4 M mannitol as osmoticum. At the eight- to ten-cell stage, divided cells were transferred to Kao’s basal medium supplemented with 0.3 M sucrose as carbon source and 0.1% agarose, 2 mg/l BA, 2 mg/l zeatin (ZEA), 1 mg/l NAA, and 0.5 mg/l Kin for callus proliferation. After 35 d, when small calli reached 2–3 mm in diameter, calli were transferred to regeneration medium containing 5 mg/l ZEA and 2 mg/l indole-3-acetic acid. The chromosome numbers of putative somatic hybrids varied from 46 to 54. A total of ten plants showed a 0.5-kb, CMS-specific band, while two regenerated plants showed a missing band similar to that of leaf mustard by polymerase chain reaction amplification using Ogura CMS-specific primers. Hybrid state was confirmed by random amplified polymorphic DNA analysis. Regenerated plants had normal petals and stamens, but only two plants produced pollen and set seed.     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

Microspore culture protocol for Indonesian <Emphasis Type="Italic">Brassica oleracea</Emphasis>

A microspore culture protocol for Brassica oleracea of Indonesian origin (cv. ‘Kemeh’) has been successfully established. A high number of embryos formed with high microspore density i.e. 15 × 10⁴ cells/ml. Embryo formation was improved by using flower buds (4.5–4.6 mm in length) as explants, a temperature treatment at 30.5°C for 48 h and then transfer to 25°C continuously until embryos formed. A total of 295 embryos were obtained from 189 buds, 30% of which were abnormal (i.e. with an abnormal cotyledon or lacking hypocotyls). All normal embryos that grew and survived, 165 in total, were successfully transferred to soil and grew well in plastic bags (15 cm in diameter) containing a mixture of burned-rice husk and organic manure (1:1, v/v).     

Mapping of a locus for adult plant resistance to downy mildew in broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> convar. <Emphasis Type="Italic">italica</Emphasis>)

The identification of the gene Pp523, conferring downy mildew resistance to adult plants of broccoli (Brassica oleracea convar. italica), led to the construction of a genetic map that included this resistance locus, 301 amplified fragment length polymorphisms, 55 random amplified polymorphic DNAs, 46 inter-simple sequence repeats, three simple sequence repeats, four other PCR markers and a flower colour locus, all gathered into nine major linkage groups. Nineteen additional molecular markers were clustered into one group of four markers, one group of three markers and six pairs of markers. The map spans over 731.9 cM, corresponding to 89.5% of the 818 cM estimated to be the total genome length. A significant number of the mapped markers, 19.3%, showed distorted segregation. The average distance between mapped adjacent markers is 1.64 cM, which places this map among the densest published to date for this species. Using bulked segregant analysis, we identified a group of molecular markers flanking and closely linked in coupling to the resistance gene and included these in the map. Two markers linked in coupling, OPK17_980 and AT.CTA_133/134, are located at 3.1 cM and 3.6 cM, respectively, at each side from the resistance gene. These markers can be used for marker-assisted selection in breeding programs aiming at the introgression of this gene in susceptible B. oleracea genotypes. The fine mapping of the genomic region surrounding the Pp523 resistance gene is currently being carried out, a basic condition for its isolation via positional cloning.     

Mapping and characterization of <Emphasis Type="Italic">FLC</Emphasis> homologs and QTL analysis of flowering time in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

The FLC gene product is an inhibitor of flowering in Arabidopsis. FLC homologs in Brassica species are thought to control vernalization. We cloned four FLC homologs (BoFLCs) from Brassica oleracea. Three of these, BoFLC1, BoFLC3 and BoFLC5, have been previously characterized. The fourth novel sequence displayed 98% sequence homology to the previously identified gene BoFLC4, but also showed 91% homology to BrFLC2 from Brassica rapa. Phylogenetic analysis showed that this clone belongs to the FLC2 clade. Therefore, we designated this gene BoFLC2. Based on the segregation of RFLP, SRAP, CAPS, SSR and AFLP loci, a detailed linkage map of B. oleracea was constructed in the F₂ progeny obtained from a cross of B. oleracea cv. Green Comet (broccoli; non-vernalization type) and B. oleracea cv. Reiho (cabbage; vernalization type), which covered 540 cM, 9 major linkage groups. Six quantitative trait loci (QTL) controlling flowering time were detected. BoFLC1, BoFLC3 and BoFLC5 were not linked to the QTLs controlling flowering time. However, the largest QTL effect was located in the region where BoFLC2 was mapped. Genotyping of F₂ plants at the BoFLC2 locus showed that most of the early flowering plants were homozygotes of BoFLC-GC, whereas most of the late- and non-flowering plants were homozygotes of BoFLC-Rei. The BoFLC2 homologs present in plants of the non-vernalization type were non-functional, due to a frameshift in exon 4. Moreover, duplications and deletions of BoFLC2 were detected in broccoli and a rapid cycling line, respectively. These results suggest that BoFLC2 contributes to the control of flowering time in B. oleracea.     

Genetics and fine mapping of a yellow-green leaf gene (<Emphasis Type="Italic">ygl</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis> L.)

Cabbage (Brassica oleracea var. capitata L.) is one of the most popular cultivated vegetables worldwide. Cabbage has rich phenotypic diversity, including plant height, head shape, head color, leaf shape and leaf color. Leaf color plays an important role in cabbage growth and development. At present, there are few reports on fine mapping of leaf color mutants in B. oleracea. In this study, a naturally occurring yellow-green leaf cabbage mutant (YL-1), derived from the self-pollinated progenies of the hybrid ‘Hosom’, was used for inheritance analysis and gene mapping. Segregation populations including F₂ and BC₁ were generated from the cross of two inbred lines, YL-1 and 01–20. Genetic analysis with the F₂ and BC₁ populations demonstrated that the yellow-green leaf color was controlled by a single recessive nuclear gene, ygl-1. Insertion–deletion (InDel) markers, designed based on the parental re-sequencing data, were used for the preliminary mapping with BSA (bulked segregant analysis) method. A genetic map constructed with 15 InDels indicated that ygl-1 was located on chromosome C01. The ygl-1 gene is flanked by InDel markers ID2 and M8, with genetic distances of 0.4 cM and 0.35 cM, respectively. The interval distance between two markers is 167 kb. Thus, it enables us to locate the ygl-1 gene for the first time in B. oleracea. This study lays the foundation for candidate gene prediction and ygl-1gene cloning.     

Acclimation of photosynthesis to temperature in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Plants differ in how much the response of net photosynthetic rate (P _N) to temperature (T) changes with the T during leaf development, and also in the biochemical basis of such changes in response. The amount of photosynthetic acclimation to T and the components of the photosynthetic system involved were compared in Arabidopsis thaliana and Brassica oleracea to determine how well A. thaliana might serve as a model organism to study the process of photosynthetic acclimation to T. Responses of single-leaf gas exchange and chlorophyll fluorescence to CO₂ concentration measured over the range of 10–35 °C for both species grown at 15, 21, and 27 °C were used to determine the T dependencies of maximum rates of carboxylation (V_Cmax), photosynthetic electron transport (J_max), triose phosphate utilization rate (TPU), and mesophyll conductance to carbon dioxide (g’_m). In A. thaliana, the optimum T of P _N at air concentrations of CO₂ was unaffected by this range of growth T, and the T dependencies of V_Cmax, J_max, and g’_m were also unaffected by growth T. There was no evidence of TPU limitation of P _N in this species over the range of measurement conditions. In contrast, the optimum T of P _N increased with growth T in B. oleracea, and the T dependencies of V_Cmax, J_max, and g’_m, as well as the T at which TPU limited P _N all varied significantly with growth T. Thus B. oleracea had much a larger capacity to acclimate photosynthetically to moderate T than did A. thaliana.     

Fine mapping of a male sterility gene <Emphasis Type="Italic">MS-cd1</Emphasis> in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

A dominant male sterility (DGMS) line 79-399-3, developed from a spontaneous mutation in Brassica oleracea var. capitata, has been widely used in production of hybrid cultivars in China. In this line, male sterility is controlled by a dominant gene Ms-cd1. In the present study, fine mapping of Ms-cd1 was conducted by screening a segregating population Ms79-07 with 2,028 individuals developed by four times backcrossing using a male sterile Brassica oleracea var. italica line harboring Ms-cd1 as donor and Brassica oleracea var. alboglabra as the recipient. Bulked segregation analysis (BSA) was performed for the BC₄ population Ms79-07 using 26,417 SRAP primer SRAPs and 1,300 SSRs regarding of male sterility and fertility. A high-resolution map surrounding Ms-cd1 was constructed with 14 SRAPs and one SSR. The SSR marker 8C0909 was closely linked to the MS-cd1 gene with a distance of 2.06 cM. Fourteen SRAPs closely linked to the target gene were identified; the closest ones on each side were 0.18 cM and 2.16 cM from Ms-cd1. Three of these SRAPs were successfully converted to dominant SCAR markers with a distance to the Ms-cd1 gene of 0.18, 0.39 and 4.23 cM, respectively. BLAST analysis with these SCAR marker sequences identified a collinear genomic region about 600 kb in scaffold 000010 on chromosomeA10 in B. rapa and on chromosome 5 in A. thaliana. These results provide additional information for map-based cloning of the Ms-cd1 gene and will be helpful for marker-assisted selection (MAS).     

Induction and origin of adventitious roots from chimeras of <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Adventitious roots were induced from shoots and leaves of the chimera plant TCC (LI-LII-LIII = TCC; T = Tuber mustard, C = Red Cabbage), previously developed by in vitro grafting of tuber mustard (Brassica juncea) and red cabbage (B. oleracea). The regeneration frequency of adventitious roots from TCC shoots and leaf sections was markedly higher than that obtained from the parents TTT (tuber mustard) and CCC (red cabbage). Moreover, levels of α-naphthaleneacetic acid in the culture medium had lower effects on rooting efficiency of TCC chimeras compared to those of TTT and CCC. The number and fresh weight of adventitious roots per TCC shoot, 13.11 roots and 0.274 g, respectively, were also significantly higher than those of the parents. This demonstrated that replacing the histogenic LI layer (the outermost apical cell layer) with a different genotype might improve adventitious root induction capability of these vegetative tissues due to likely synergistic effects between LI and the other two histogenic layers, LII and LIII. Following polymerase chain reaction analysis and histological investigation, it was found that these adventitious roots originated from the LIII histogenic layer.     

Induction and origin of adventitious shoots from chimeras of <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

The chimeras between tuber mustard (Brassica juncea) and red cabbage (B. oleracea) were artificially synthesized in our previous study. Adventitious shoots were induced from nodal segments and leaf discs of TCC (LI-LII-LIII, LI -the outmost layer of shoot apical meristem; LII -the middle layer; LIII -the innermost layer. T = Tuber mustard, C = Red cabbage) chimeras. The origin of the shoots was analyzed by histology and molecular biology. As a result, the frequency of adventitious shoot induction rose with the increase of BA in MS medium in the area of the nodes. However, there was no different induction frequency of adventitious shoots from nodal segment bases in media with different BA concentrations. Most adventitious shoots (clustered shoots) arising from the node area were TTT (Tuber mustard- Tuber mustard- Tuber mustard) and only 4 shoots were chimeras, which indicated that more shoots originated from LI than from LII and LIII. All shoots from nodal segment bases were CCC (Red cabbage-Red cabbage- Red cabbage), indicating that the shoots originated from LII or LII and LIII. There were significant differences in the regeneration rate in the margin of the leaf discs among the three combinations of BA and NAA. Most adventitious shoots from the margin of leaf discs were CCC but 2 out of 70 were chimeras, which indicated that more shoots originated from LII or LII and LIII than from LI. All chimeras obtained by regeneration were different from the original explant donor in type in the present study. The origin of the adventitious shoots varied with the site of origin on the donor plant, and could be multicellular and multihistogenic.     

Mapping of days to flower and seed yield in spring oilseed <Emphasis Type="Italic">Brassica napus</Emphasis> carrying genome content introgressed from <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Earliness of flowering and maturity and high seed yield are important objectives of breeding spring Brassica napus canola. Previously, we have introgressed earliness of flowering from Brassica oleracea into spring B. napus canola through interspecific crossing between these two species. In this paper, we report quantitative trait locus (QTL) mapping of days to flower and seed yield by use of publicly available markers and markers designed based on flowering time genes and a doubled haploid population, derived from crossing of the spring canola parent and an early flowering line developed from a B. napus × B. oleracea cross, tested in nine field trials for over 5 years. Five genomic regions associated with days to flower were identified on C1, C2, C3, and C6 of which the single QTL of C1 was detected in all trials; in all cases, the allele introgressed from B. oleracea reduced the number of days to flower. BLASTn search in the Brassica genomes located the physical position of the QTL markers and identified putative flowering time genes in these regions. In the case of seed yield, ten QTL from eight linkage groups were detected; however, none could be consistently detected in all trials. The QTL region of C1 associated with days to flower did not show significant association with seed yield in more than 80% of the field trials; however, in a single trial, the allele introgressed from B. oleracea exerted a negative effect on seed yield. Thus, the genomic regions and molecular markers identified in this research could potentially be used in breeding for the development of early flowering B. napus canola cultivars without affecting seed yield in a majority of the environments.     

Genetic analysis of<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> susceptibility in<Emphasis Type="Italic"> Brassica oleracea</Emphasis>

The genetic control and heritability of Agrobacterium tumefaciens susceptibility was investigated using a doubled haploid (DH) mapping population of Brassica oleracea and the associated RFLP map. Preliminary studies were carried out by analysis of an 8×8 diallel, for which the parental lines were selected to include a range of susceptibilities to A. tumefaciens. The variation observed within the diallel was attributed to both additive and dominant gene effects, with additive gene effects being more important. A broad sense heritability value of 0.95 suggested that 95% of the observed variation was due to genetic effects, with just 5% attributed to non-genetic or environmental effects. A high narrow-sense heritibility value of 0.79 suggested that 79% of this trait was controlled by additive gene effects and, therefore, the potential to introduce this trait into breeding material is high. Fifty-nine DH lines from the mapping population were screened for susceptibility towards A. tumefaciens. Variation in susceptibility was observed across the population. The results of the DH screen were entered into the mapping programme MAPQTL and a highly significant quantitative trait loci (QTL) associated with susceptibility to A. tumefaciens was identified on linkage group 09. The use of substitution lines covering this region confirmed the location of this QTL. This work shows that susceptibility to A. tumefaciens is a heritable trait, and the transfer of susceptibility into resistant lines is demonstrated. These findings may help to overcome genotype restrictions to genetic transformation.Communicated by G. Wenzel     

Detection and resolution of genetic loci affecting circadian period in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Circadian rhythms regulate many aspects of plant growth, fitness and vigour. The components and detailed mechanism of circadian regulation to date have been dissected in the reference species Arabidopsis thaliana. To determine the genetic basis and range of natural allelic variation for intrinsic circadian period in the closest crop relatives, we used an accurate and high throughput data capture system to record rhythmic cotyledon movement in two immortal segregating populations of Brassica oleracea, derived from parent lines representing different crop types. Periods varied between 24.4 and 26.1 h between the parent lines, with transgressive segregation between extreme recombinant lines in both populations of ∼3.5 h. The additive effect of individual QTL identified in each population varied from 0.17 to 0.36 h. QTL detected in one doubled haploid population were verified and the mapping intervals further resolved by determining circadian period in genomic substitution lines derived from the parental lines. Comparative genomic analysis based on collinearity between Brassica and Arabidopsis also allowed identification of candidate orthologous genes known to regulate period in Arabidopsis, that may account for the additive circadian effects of specific QTL. The distinct QTL positions detected in the two populations, and the extent of transgressive segregation suggest that there is likely to be considerable scope for modulating the range of available circadian periods in natural populations and crop species of Brassica. This may provide adaptive advantage for optimising growth and development in different latitudes, seasons or climate conditions.     

Tissue-culture enhanced transposition of the maize transposable element <Emphasis Type="Italic">Dissociation</Emphasis> in <Emphasis Type="Italic">Brassica oleracea</Emphasis> var. '<Emphasis Type="Italic">Italica</Emphasis>'

To investigate the potential of heterologous transposons as a gene tagging system in broccoli (Brassica oleracea var. Italica), we have introduced a Ds-based two-element transposon system. Ds has been cloned into a 35S-SPT excision-marker system, with transposition being driven by an independent 35S-transposase gene construct (Tpase). In three successive selfed generations of plants there was no evidence of germinal-excision events. To overcome this apparent inability to produce B. oleracea plants with germinal excisions, we performed a novel tissue-culture technique to select for fully green shoots from seed with somatic-excision events. The results showed a very high efficiency of regeneration of fully green plants (up to 65%) and molecular analysis indicated that the plants genetically were like plants that contain a germinal-excision event. Further molecular analysis of these plants showed that 69% exhibited reinsertion of Ds back into the plant genome. Sequencing of donor-site footprints after Ds excision, revealed that there is an indication of more-severe deletions and rearrangements when higher concentrations of streptomycin are used in the tissue-culture selection process. Adapted versions of this regeneration technique have a high potential for providing germinal excision-like events in heterologous plants species which show low transposon activity. Alternatively, there is the potential to increase the proportion of 'germinal' plants in earlier generations of more-active plant species.     

Map-based cloning of the dominant genic male sterile <Emphasis Type="Italic">Ms</Emphasis>-<Emphasis Type="Italic">cd1</Emphasis> gene in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

Key message

Using map-based cloning, we delimited the Ms - cd1 gene responsible for the male sterile phenotype in B. oleracea to an approximately 39-kb fragment. Expression analysis suggests that a new predicted gene, a homolog of the Arabidopsis SIED1 gene, is a potential candidate gene.

Abstract

A dominant genic male sterile (DGMS) mutant 79-399-3 in Brassica oleracea (B. oleracea) is controlled by a single gene named Ms-cd1, which was genetically mapped on chromosome C09. The derived DGMS lines of 79-399-3 have been successfully applied in hybrid cabbage breeding and commercial hybrid seed production of several B. oleracea cultivars in China. However, the Ms-cd1 gene responsible for the DGMS has not been identified, and the molecular basis of the DGMS is unclear, which then limits its widespread application in hybrid cabbage seed production. In the present study, a large BC₉ population with 12,269 individuals was developed for map-based cloning of the Ms-cd1 gene, and Ms-cd1 was mapped to a 39.4-kb DNA fragment between two InDel markers, InDel14 and InDel24. Four genes were identified in this region, including two annotated genes based on the available B. oleracea annotation database and two new predicted open reading frames (ORFs). Finally, a newly predicted ORF designated Bol357N3 was identified as the candidate of the Ms-cd1 gene. These results will be useful to reveal the molecular mechanism of the DGMS and develop more practical DGMS lines with stable male sterility for hybrid seed production in cabbage.

     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Production and analysis of intergeneric somatic hybrids between <Emphasis Type="Italic">Brassica oleracea</Emphasis> and<Emphasis Type="Italic"> Matthiola incana</Emphasis>

The gene pool of Brassica oleracea was enriched via intergeneric somatic hybridization between B. oleracea (2n = 18) and Matthiola incana (2n = 14). One hundred and eighteen plants were obtained from 96 calli. Only four plants (H1, H2, H3 and H4) showed an intermediate phenotype from the parents; among these, H1 and H3 arose from the same callus. Random amplified polymorphic DNA (RAPD), sequence-related amplified polymorphism (SRAP), and cytological analyses confirmed that H1 and H3 were hybrids. The nuclear DNA content of the regenerated plants was determined by flow cytometry. More than half of the plants contained a nuclear DNA content of 1.3 to 3.9 pg/cell, which was higher than the content of B. oleracea but lower than that of M. incana. H1 contained 4.89 ± 0.02 pg of DNA per cell, while H3 nuclear DNA content was estimated at 4.87 ± 0.06 pg/cell. Cytological study of the root-tip cells revealed that the majority of the H1 and H3 hybrid cells contained 28 chromosomes.