Inheritance of isozymes in Brassica campestris L. and genetic divergence among different species of Brassiceae

Electrophoretic investigations of Brassica campestris revealed 8 polymorphic loci that had not been described earlier. The inheritance of 7 of them was determined by crosses between different accessions of B. campestris. A phylogeny of B. campestris, B. nigra, B. alboglabra, Sinapis arvensis and S. alba was estimated from the analysis of 15 isozyme loci. The phylogeny showed a close relationship between B. nigra and S. arvensis and a less close relationship between B. campestris and B. oleracea. S. alba was remotely-related to all of the other species.     

Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype

Summary An SLG gene derived from the S-locus and encoding and S-locus-specific glycoprotein of Brassica campestris L. was introduced via Agrobacterium-mediated transformation into B. oleracea L. A self-incompatible hybrid and another with partial self-compatibility were used as recipients. The transgenic plants were altered in their pollen-stigma interaction and were fully compatible upon self-pollination. Reciprocal crosses between the transgenic plants and untransformed control plants indicated that the stigma reaction was changed in one recipient strain while the pollen reaction was altered in the other. Due to interspecific incompatibility, we could not demonstrate whether or not the introduced SLG gene confers a new allelic specificity in the transgenic plants. Our results show that the introduced SLG gene perturbs the self-incompatibility phenotype of stigma and pollen.     

Regeneration of transgenic vegetable brassicas (Brassica oleracea andB. campestris) via Ri-mediated transformation

A procedure for the production of fertile transgenic brassicas via Ri-mediated transformation is reported in this paper. Transgenic hairy root lines were selected for 12 vegetable brassica cultivars and lines representing six varieties: broccoli, Brussels sprouts, cabbage, cauliflower, rapid-cycling (allBrassica oleracea) and Chinese cabbage (B. campestris). Leaf explants or petioles of intact cotyledons were co-cultivated withAgrobacterium strain A4T harbouring various binary vectors. The T-DNA region of all binary vectors contained a neomycin phosphotransferase II gene for kanamycin resistance, in addition to other genes. Hairy root lines grew prolifically on hormone-free medium containing kanamycin. Transgenic shoots were regenerated from all cultivars either spontaneously or after transfer of hairy roots to a hormone-containing medium. Southern analysis confirmed that the plants were transgenic. Plants from all brassica types were successfully transferred to greenhouse conditions. Plants were fertile and segregation analysis confirmed transmission of traits to progeny.Abbreviations BA 6-Benzylaminopurine - GUS -Glucuronidase - LS Linsmaier and Skoog medium - NAA I-Naphthaleneacetic acid - NPTII Neomycin phosphotransferase II - TDZ thidiazuron     

Genetic analysis of morphological variation in Brassica oleracea using molecular markers

A cross between the open-pollinated Brassica oleracea cabbage cultivar Wisconsin Golden Acre and the hybrid broccoli cultivar Packman was used with molecular markers to investigate the genetic control of morphological variation. Twenty-two traits derived from leaf, stem, and flowering measurements were analyzed in 90 F₂ individuals that were also classified for genotype by restriction fragment length polymorphism (RFLP) markers. Seventy-two RFLP loci, which covered the mapped genome at an average of 10 map-unit intervals on all nine linkage groups, were tested individually for associations to phenotypic measurements by single factor ANOVA, and markers with significant associations (P<0.05) were used to develop multilocus models. These data were utilized to describe the location, parental contribution of alleles, magnitude of effect, and the gene action of trait loci. Single marker loci that were significantly associated (P<0.05) with trait measurements accounted for 6.7–42.7% of the phenotypic variation. Multilocus models described as much as 60.1% of the phenotypic variation for a given trait. In some cases, different related traits had common marker-locus associations with similar gene action and genotypic class ranking. The numbers, action, and linkages, of genes controlling traits estimated with marker loci in this population corresponded to estimates based on classical genetic methods from other studies using similar, or similarly-wide, crosses. There was no evidence that genome duplication accounted for a significant portion of multiple genes controlling trait loci over the entire genome, but possible duplications of trait loci were identified for two regions with linked, duplicated marker loci.     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Molecular marker analysis of genes controlling morphological variation in Brassica rapa (syn. campestris)

Construction of a detailed RFLP linkage map of B. rapa (syn. campestris) made it possible, for the first time, to study individual genes controlling quantitative traits in this species. Ninety-five F₂ individuals from a cross of Chinese cabbage cv Michihili by Spring broccoli were analyzed for segregation at 220 RFLP loci and for variation in leaf, stem, and flowering characteristics. The number, location, and magnitude of genes underlying 28 traits were determined by using an interval mapping method. Zero to five putative quantitative trait loci (QTL) were detected for each of the traits examined. There were unequal gene effects on the expression of many traits, and the inheritance patterns of traits ranged from those controlled by a single major gene plus minor genes to those controlled by polygenes with small and similar effects. The effect of marker locus density on detection of QTL was analyzed, and the results showed that the number of QTL detected did not change when the number of marker loci used for QTL mapping was decreased from 220 to 126; however, a further reduction from 126 to 56 caused more than 15% loss of the total QTL detected. The detection of putative minor QTL by removing the masking effects of major QTL was explored.     

Secretory tapetum of Brassica oleracea L.: polarity and ultrastructural features

Summary The ultrastructure of the secretory, binucleate tapetum of Brassica oleracea in the micro spore mother cell (MMC) stage through to the mature pollen stage is reported. The tapetal cells differentiate as highly specialized cells whose development is involved in lipid accumulation in their final stage. They start breaking down just before anther dehiscence. Nuclei with dispersed chromatin, large nucleoli and many ribosomes in the cytoplasm characterize the tapetal cells. The wall-bearing tapetum phase ends at the tetrade stage. The dissolution of tapetal walls begins from the inner tangential wall oriented towards the loculus and proceeds gradually along the radial walls to the outer tangential one. The plasmodesmata transversing the radial walls between tapetal cells persist until the mature microspore, long after loss of the inner tangential wall. After wall dissolution, the tapetal protoplasts retain their integrity and position within the anther locule. The tapetal cell membrane is in direct contact with the exine of the microspores/pollen grains and forms tubular evaginations that increase its surface area and appear to be involved in the translocation of solutes from the tapetal cells to the microspores/ pollen grains. The tapetal cells exhibit a polarity expressed by spatial differentiation in the radial direction.     

The inheritance of partial self-compatibility in Brassica oleracea L. inbreds homozygous for different S-alleles

Summary In a study of partial self-compatibility in Brassica oleracea, flower number, seeded siliqua and seed production were recorded on self and cross-pollinated inflorescences of 32 progenies obtained by inter-crossing and selfing 8 plants homozygous for the incompatibility alleles S₂, S₅, S₁₅ and S₄₅.Progeny differences for both self-and out-cross seed production could be largely attributed to G.C.A. effects which were essentially uncorrelated. For cross-pollinated inflorescences heterosis was also important. Significant differences were found for selfed seed set and its two components, the proportion of flowers producing seeded siliquae and the numbers of seed per seeded siliqua, between parents with the same S-allele which could not be attributed to S-genotype alone. No evidence of increased self-compatibility in particular S-allele heterozygotes (mutual weakening) could be found.Outcross seed production depended primarily on the numbers of seeds set per seeded siliqua while self seed production was largely determined by the proportion of flowers which produced seeded siliqua. It is suggested that this is a key character for the production of inbred lines with reduced partial self-compatibility.     

Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes

Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

Synthesis and phosphorylation of pollen proteins during the pollen-stigma interaction in self-compatible Brassica napus L. and self-incompatible Brassica oleracea L.

A technique is described which permits the in vivo study of protein synthesis and phosphorylation in the pollen of Brassica spp. during the early stages of the pollen-stigma interaction. In Brassica napus and B. oleracea, compatible pollination is followed by a dramatic activation of protein synthesis in the pollen involving the synthesis of approximately 40 proteins. After incompatible pollinations in B. oleracea, virtually no newly synthesised polypeptides were detected in the pollen except for a small group of high molecular weight proteins which were not normally synthesised during compatible pollinations. Both compatible and incompatible pollinations were followed by the appearance of newly phosphorylated proteins in the pollen; these fell into four distinct groups. In B. oleracea, the number of phosphorylated proteins and the degree of phosphorylation of individual proteins within the four groups differed between compatible and incompatible pollinations. One group of phosphorylated proteins appeared to correspond with the small group of high molecular weight polypeptides which were synthesised in pollen after incompatible pollinations. These findings are discussed in the perspective of cell signalling during the pollen-stigma interaction in Brassica and also in terms of their possible implication in sporophytic self-incompatibility.     

Bioassembly of acyl lipids in microspore-derived embryos of Brassica campestris L.

The native lipid composition and the capacity of cell-free extracts to biosynthesize acyl lipids in vitro were determined for the first time using the recently reported microspore-derived (MD) embryo system from the Brassica campestris low erucic acid line BC-2 (Baillie et al. 1992). The total lipid fraction isolated from midcotyledonary stage MD embryos (21 days in culture) was composed primarily of triacylglycerol (76%) with an acyl composition quite similar to that of mature BC-2 seed. When incubated in the presence of glycerol-3-phosphate, ¹⁴C 181-CoA, and reducing equivalents, homogenates prepared from 21-day cultured MD embryos were able to biosynthesize glycerolipids via the Kennedy pathway. The maximum in vitro rate of triacylglycerol biosynthesis could more than account for the known rate of lipid accumulation in vivo. The homogenate catalyzed the desaturation of 181 to 182 and to a lesser extent, 183. The newly-synthesized polyunsaturated fatty acids initially accumulated in the polar lipid fraction (primarily phosphatidic acid and phosphatidylcholine) but began to appear in the triacylglycerol fraction after longer incubation periods. As expected for a low erucic acid cultivar, homogenates of MD embryos from the BC-2 line were incapable of biosynthesizing very long chain monounsaturated fatty acyl moieties (201 and 221) from 181-CoA in vitro. Nonetheless, embryo extracts were still capable of incorporating these fatty acyl moieties into triacylglycerols when supplied with ¹⁴C 201-CoA or ¹⁴C 221-CoA. Collectively, the data suggest that developing BC-2 MD embryos constitute an excellent experimental system for studying pathways for glycerolipid bioassembly and the manipulation of this process in B. campestris.Abbreviations CPT sn-1,2-diacylglycerol cholinephosphotransferase - DAG diacylglycerol - DGAT diacylglycerol acyltransferase - DGDG digalactosyldiacylglycerol - G-3-P glycerol-3-phosphate - G-3-PAT glycerol-3-phosphate acyltransferase - LPA lyso-phosphatidic acid - LPAT lyso-phosphatidic acid acyltransferase - LPC lyso-phosphatidylcholine - LPCAT acyl-CoA: lyso-phosphatidylcholine acyltransferase - LPE lyso-phosphatidylethanolamine - MGDG monogalactosyldiacylglycerol - PA phosphatidic acid - PA Phosphatase, phosphatidic acid phosphatase - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - TAG triacylglycerol - 181-CoA oleoyl-Coenzyme A - 181 oleic acid, cis-9-octadecenoic acid - 182 linoleic acid, cis-9,12-octadecadienoic acid - 183 -linolenic acid, cis-9,12,15-octadecatrienoic acid - 201 cis-11-eicosenoic acid - 221 erucic acid, cis-13-docosenoic acid; all other fatty acids are designated by number of carbon atoms: number of double bonds National Research Council of Canada Publication No. 35896     

Plant regeneration from cotyledonary protoplasts of cauliflower (Brassica oleracea L. var.botrytis L.)

Summary Protoplasts isolated enzymatically from precultured cotyledonary leaves ofB. oleracea var.botrytis and cultured in KM8p medium (Kao andMichayluk 1975) underwent sustained divisions in about 0.1% population to eventually produce callus, whereas mesophyll protoplasts from either field grown orin vitro raised plants failed to divide. The callus readily differentiated on Murashige-Skoog medium as modified for shoot culture (Binding 1974) to give rise to shoot and roots.     

Genetic analysis of novel intra-species unilateral incompatibility in Brassica rapa (syn. campestris) L.

Plants have evolved many systems to prevent inappropriate fertilization. Among them, incompatibility is a well-organized system in which pollen germination or pollen-tube growth is inhibited in pistils. Self-incompatibility (SI), rejecting self-pollen, promotes outbreeding in flowering plants. On the other hand, inter-species incompatibility, preventing gene flow among species to restrict outbreeding, usually occurs unilaterally, and is known as unilateral incompatibility (UI). In Brassicaceae, little is known about the molecular mechanism of UI, although S-locus genes involved in recognition of self-pollen have been characterized in the SI system. In the present study, we characterized novel UI observed between members of the same species, Brassica rapa; pollen of Turkish SI lines was specifically rejected by pistils of the Japanese commercial SI variety Osome. The incompatible phenotype of this intra-species UI closely resembled that of SI. Segregation analysis revealed that the pollen factor of this UI was not linked to the S-locus.The revised version was published online in December 2004 with corrections to figure 1.     

Cytological study of interspecific hybrid between Brassica campestris x B. hirta (Sinapis alba)

Interspecific hybrids from the crossing Brassica campestris x B. hirta are reported in our study for the first time. F₁ plants were obtained by using ovary culture. The phenotype of hybrids was similar to B. napus; the plants were self-fertile. Investigation of meiotic division and nuclear DNA content measurements showed the amphidiploid origin of these hybrids. The relationship between genome A and D, as well as the spontaneous amphidiploidization of the hybrids, are discussed.     

Male sterility caused by cytoplasm of Brassica oxyrrhina in B. campestris and B. juncea

Summary Synthetic alloploid Brassica oxyrrhina (2n = 18, OO) x B. campestris (2n = 20, AA) was repeatedly backcrossed with B. campestris to place B. campestris nucleus in the cytoplasm of B. oxyrrhina. Alloplasmic plants, obtained in BC₅ generation, were stably male sterile but mildly chlorotic during initial development. Synthetic alloploid B. oxyrrhina-campestris was also hybridized with B. juncea to transfer B. oxyrrhina cytoplasm. Segregation for green and chlorotic plants was observed in BC₁ and BC₂ generations. By selection, however, normal green male sterile B. juncea was obtained in BC₃. Pollen abortion in both B. campestris and B. juncea is post-meiotic.     

Generation of a Brassica oleracea composite RFLP map: linkage arrangements among various populations and evolutionary implications

Summary A composite linkage map of Brassica oleracea was developed from maps of four different populations, derived from 108 DNA, isozyme and morphological loci covering over 747 centimorgans in 11 linkage groups. Of these linkage groups, 8 were assigned to their respective chromosomes by alignment with gene synteny groups of B. oleracea. Distortions in segregation ratios increased with the level of divergence of the parents and were attributed to differentiation of parental chromosomes. Comparison of the individual maps demonstrates that the B. oleracea genome undergoes frequent chromosomal rearrangement, even at the subspecies level. Small inversions were the most frequent form of aberration followed by translocations. The former type of aberration could occur without a noticeable effect on meiotic behavior of chromosomes or on pollen fertility. The obvious deduction from the composite map is that a large fraction of the B. oleracea genome is duplicated, falling into three classes: randomly dispersed, linked-gene families, and blocks duplicated in non-homologous chromosomes. The genealogy of chromosomes sharing duplicated segments was formulated and indicates that B. oleracea is a secondary polyploid species derived from ancestral genome(s) of fewer chromosomes.     

Transformation of cauliflower (Brassica oleracea L. var. botrytis) — an experimental survey

The paper compares different approaches for the genetic transformation of cauliflower (Agrobacterium-mediated, PEG-mediated and/or electroporation). Transient expression of the neomycin phosphotransferase II (NPTII) gene could be detected after direct gene transfer. Stable transformation was achieved using both Agrobacterium-mediated and direct gene transfer. Expression as well as incorporation of the NPTII sequence could be demonstrated.     

The impact of domestication on distribution of allozyme variation within and among cultivars of radish,Raphanus sativus L.

Summary Allozyme surveys of cultivated plant species generally report little within-cultivar variation, but considerable among-cultivar variation. This trend contrasts with natural plant populations in which most allozyme variation resides within, rather than among, populations. The difference may be an artifact of the extreme inbreeding techniques used to develop and propagate these crops, rather than a consequence of domestication per se. To test this hypothesis, we compared the population genetic structure of 24 lines of radish cultivars — a domesticated species developed and maintained as open-pollinated, outcrossed populations — with four wild radish populations in California. Although the wild populations displayed more overall allozyme variation than the cultivars, most of the allozyme variation in the cultivars remains partitioned within, rather than among, lines. Apparently, how a crop is developed and maintained can have a profound influence on the organization of genetic variation of that species.