Production of intergeneric hybrids between a C3-C4 intermediate species Moricandia arvensis and a C3 species Brassica oleracea through ovary culture

Summary Intergeneric hybrids between Moricandia arvensis (a C₃-C₄ intermediate species) and Brassica oleracea (a C₃ species) were obtained through ovary culture. Many hybrid embryos (2.71 per pollination) were produced in the M. arvensis × B. oleracea cross, but none were produced from the reciprocal cross. Though most embryos failed to develop into plantlets directly, plants were obtained by inducing shoots from hypocotyl explants. The hybrid plants were morphologically intermediate between the parents except for the petal color. Cytogenetic observations indicated that partial homology existed between the two genomes. Ovary culture is an efficient technique for gene transfer from M. arvensis to B. oleracea.     

Moricandia arvensis cytoplasm based system of cytoplasmic male sterility in Brassica juncea: Reappraisal of fertility restoration and agronomic potential

Cytoplasmic male sterility (CMS) system based on the cytoplasm from Moricandia arvensis (mori) was investigated for fertility restoration and agronomic potential. Fertility restorer gene for mori CMS was introgressed from cytoplasm donor species as all the evaluated Brassica juncea genotypes (155) acted as sterility maintainers. The allosyndetic pairing between M^a and the A/B genome chromosomes in the monosomic addition plants (2n= 18II + 1M^a) facilitated the gene introgression. Partial fertility restoration (43–52% pollen grain stainability) in F₁ hybrids and absence of segregation for male sterility in F₂ progenies suggested gametophytic control of fertility restoration. The pollen fertility in the F₁ hybrids was, however, sufficient to ensure complete seed set upon bag selfing. Introgression from M. arvensis also helped in correction of chlorosis associated with mori cytoplasm in CMS and fertile alloplasmic B. juncea plants. Yield evaluation of thirty F₁ hybrids having the same nuclear genotype but varied male sterilizing cytoplasms (mori, oxy, lyr, refined ogu), in comparison to respective euplasmic hand bred control hybrids, allowed an estimate of yield penalty associated with different CMS systems. It ranged from 1.8% to 61.6%. Hybrids based on cytoplasmically refined ogu were most productive followed by those based on cytoplasmically refined mori CMS. The male sterility systems emanating from somatic hybridization were found superior than those developed from sexual hybridization.     

Successful backcrosses of somatic hybrids between Sinapis alba and Brassica oleracea with the Brassica oleracea parent

Somatic hybrids between Sinapis alba (2n= 24) and Brassica oleracea (2n= 18) have been backcrossed with the B. oleracea parent. Whereas backcrosses with the diploid B. oleracea parent were unsuccessful, 344 BC₁ seeds could be obtained from inter-valence crosses with tetraploid B. oleracea (2n= 4x= 36). The investigated 96 BC₁ plants segregated for morphological traits and for fertility. They were backcrossed with diploid B. oleracea or self-pollinated, depending on their male fertility. The BC₁F₂ and BC₂ progenies segregated well for the morphological traits. Disturbances were observed especially in the generative phase (flower development and pollen fertility). Both male fertile and male sterile BC₁F₂ and BC₂ plants were obtained and backcrossed or self-pollinated with the B. oleracea parent. The presence of either one of the parental or the cybrid organelle genomes was detected. In the progenies, a stable maternal inheritance of the organelle genome patterns was observed. Isozyme analyses revealed polymorphism for the leucine aminopeptidase (LAP) which was used for the identification of S. alba genes in the progenies. Cytological investigations showed a clear differentiation between the BC₁F₂ and BC₂ plants. Whereas the BC₁F₂ plants possess large numbers of chromosomes ranging from 34 to 40, the BC₂ material was strongly reduced to chromosome numbers ranging from 20 to 22. Preliminary investigation of the meiosis suggests the possibility of introgressions of S. alba-DNA into the B. oleracea genome.     

Production and characterization of intergeneric hybrids between Brassica oleracea and a wild relative Moricandia arvensis

Intergeneric crosses were made between Brassica oleracea and Moricandia arvensis utilizing embryo rescue. Six F₁ hybrid plants were generated in the cross‐combination of B. oleracea × M. arvensis from 64 pods by the placenta‐embryo culture technique, whereas three plants were produced in the reciprocal cross from 40 pods by the ovary culture technique. The hybrid plants were ascertained to be amphihaploid with 2n = 23 chromosomes in mitosis and a meiotic chromosome association of (0–3)II + (17–23)I at metaphase I (M I). In the backcross with B. oleracea, some of these hybrids developed sesquidiploid BC₁ plants with 2n = 32 chromosomes that predominantly exhibited a meiotic configuration of (9II + 14I) in pollen mother cells. The following backcross of BC₂ plants to B. oleracea generated 48 BC₃ progeny with somatic chromosomes from 2n = 19 to 2n = 41. The 2n = 19 plants showed a chromosomal association type of (9II + 1I) and a chromosomal distribution type of (9¹/₂ + 9¹/₂) or (9 + 10) at M I and M II, respectively. These facts might suggest that they were monosomic addition lines (MALs) of B. oleracea carrying a single chromosome of M. arvensis that could offer potential for future genetic and breeding research, together with other novel hybrid progeny developed in this intergeneric hybridization.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. II. Morphology,fertility and cytology of the F1 hybrid

Summary F₁ hybrids of triazine resistant Brassica napus and triazine susceptible B. oleracea were morphologically intermediate to the parent species. Of 49 hybrids examined, 44 had 28 chromosomes, two had 37, one had 38 and two had 56. The 38-chromosome plant was thought to be a matromorph, the others, A₁C₁C (28), A₁C₁CC (37) or A₁A₁C₁C₁CC (56) type hybrids. Pollen stainability averaged 9.0% in the sesquidiploid, 32.0% in the tetraploids and 89.5% in the hexaploids. All the interspecific hybrids were resistant to 1.0×10^-4 mol L^-1 atrazine. The sesquidiploid hybrids produced gametes with chromosome numbers ranging from 9 to 17 and the tetraploid hybrid gametes had chromosome numbers from 15 to 22. Most hybrids produced self-seed. The partial fertility of these hybrids may permit their backcrossing to one or both parents.     

Characterization of Somatic Brassies napus Hybrids by Polyacrylamide Gel Electrophoresis

Three somatic hybrids obtained by fusion of protoplasts from Brassica oleracea and B. campestris were analyzed by gel electrophoresis and compared with their respective parental species. By comparing multiple forms of esterases and phosphorylases it could be demonstrated that in all cases the hybrid plants contained one or more enzymes from each parent.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.     

Synthesis and sterility of raphanobrassica

Summary The synthesis of Raphanobrassica (2n=36, rrcc) from Raphanus sativus (2n=18, rr) and Brassica oleracea (2n=18, cc) is described a) by colchicine treatment of diploid hybrids; b) by crossing autotetraploid froms of the parent species.The variation within R. sativus and B. oleracea suggests that a range of morphologically distinct Raphanobrassica forms may be created, some of which may have agronomic potential and in particular, it is hoped, Plasmodiophora resistance.Inter-generic hybrids were readily obtained from crossing the parental species at both 2x and 4x chromosome levels, but only with R. sativus as female parent.Details are given of the morphology, fertility and chromosome behaviour of both diploid F₁ R. sativus × B. oleracea hybrids and of the amphidiploid Raphanobrassica.Synthesized Raphanobrassica plants proved, in general, highly sterile. Some aneuploids resulted from 4x R. sativus × 4x B. oleracea crosses but most progeny were euploid and showed almost regular chromosome association. A number of stunted, deformed plants were obtained from both 2x and 4x crosses. Vigour, fertility and aneuploidy appeared unconnected in the amphidiploid.Previous work on Raphanobrassica is reviewed. It is concluded that the extremely low fertility encountered in the present study is more likely to be the result of genic imbalance than to cytological anomalies which appear to be of lesser significance.     

Cytogenetics of the 'glandless-seed and glanded-plant' trait from Gossypium sturtianum Willis introgressed into upland cotton (Gossypium hirsutum L.)

In order to introgress the ‘glandless-seed and glanded-plant’ trait from Gossypium sturtianum Willis (2n= 2x= 26, C₁ genome) into the cultivated upland cotton Gossypium hirsutum L. (2n= Ax= 52 (AD), genome), two trispecific hybrids have been created using either Gossypium thurberi Torado (2n= 2x= 26, D₁ genome) or Gossypium raimondii Ulbrich (2n= 2x= 26, D₅ genome) as bridge species. The cross of both trispecific hybrids by G. hirsutum produced the first backcross progenies (BCl). Cytogenetic analysis showed that the trispecific hybrids had 52 chromosomes, their chromosome configurations at metaphase I (Ml) being 15.071 + 15.3411 + 0.93III + 0.69IV + 0.26VI in G. thurberi×G. sturtianum×G. hirsutum (TSH) and 14.421 + 17.0311 + 0.82III + 0.15IV + 0.07VI in G. hirsutum × G. raimondii ×. G. sturtianum (HRS), respectively. Among six BCl plants analysed, the only plant expressing the ‘glandless-seed and glanded-plant’ trait had 52 chromosomes and a meiotic configuration of 5.261 + 20.61II + 0.69III + 0.77IV at MI. Pollen fertility was 2.90% in TSH, 8.97% in HRS, and ranged from 0% to 40.28% in the BCl progenies. The introgressed BCl plant is perennial in growth habit. It can be used in breeding programmes aiming at the introgression of the ‘glandless-seed and glanded-plant’ trait into a cultivar of upland cotton.     

Cytogenetical and molecular investigations on somatic hybrids of Sinapis alba and Brassica juncea and their backcross progeny

Somatic hybrids of Sinapis alba+Brassica juncea (Sal Sal AABB) were synthesized by protoplast electrofusion. They were true genomic allopolyploids since they possessed 60 chromosomes, i.e. the sum of S. alba (2n= 24) and B. juncea (2n= 36) chromosomes. Chromosome pairing was predominantly bivalent with the occasional occurrence of multivalents in the pollen mother cells at diakinesis and metaphase I. Hybrids were completely pollen-sterile, but produced seeds on back-crossing with B. juncea and B. campestris. A total of 37 BC₁ plants were raised from two somatic hybrids (JS-1 and JS-2) and 24 of these were analysed cytologically. The 22 plants originating from the pollinations of somatic hybrids with B. juncea showed a chromosome configuration of 18II+12I and had 42–86% pollen fertility. Two plants from the backcrosses of the somatic hybrid with B. campestris formed 10II +20I, and had 0–4% fertile pollen. Total DNA analysis by probing with pTA71 carrying a full-length 18S–25S rDNA fragment of the wheat nuclear genome revealed that the two somatic hybrids possessed all the characteristic bands of both the species, confirming their hybridity. Probing with the mitochondrial coxI and atp9 genes indicated mitochondrial genome recombination in the hybrids. Hybridization with chloroplast-specific psbD indicated that both the somatic hybrids possessed the cp genome of S. alba origin.     

Chromosomal structural changes in Capsicum annuum L. and C. chinense Jacq.

Summary Wide hybridizations between M. arvensis and Brassica amphidiploid species (B. napus and B. juncea) were carried out in order to incorporate desirable traits of M. arvensis into Brassica crops. Crossing barriers between them were present without the use of in vitro techniques. F₁ hybrids have been produced through ovary culture, when M. arvensis were used as a female parent. Higher hybrid embryo productivity (3.07 embryos per pollination) was obtained in the cross of M. arvensis x B. napus than in that of M. arvensis x B. juncea (0.79 embryos). The hybridity was confirmed by morphology, cytology, isozyme and Southern analyses. The first backcrossing progenies and open pollinated ones were produced.     

Fertile somatic hybrids of Solanum etuberosum (+) dihaploid Solanum tuberosum and their backcrossing progenies: relationships of genome dosage with tuber development and resistance to potato virus Y

The wild non-tuberous species Solanumetuberosum is resistant to biotic andabiotic stresses, but is very difficult tocross with cultivated potato. Therefore,interspecific somatic hybrids between adihaploid clone of potato S.tuberosum (2n=2x=24, AA genome) and thediploid species S. etuberosum(2n=2x=24, EE genome) were produced byprotoplast fusion. Among the 7 fertilefusion hybrids analysed by genomic insitu hybridisation (GISH), three groups ofplants were found with the genomicconstitution of AAEE, AAEEEE and AAAAEE.Four fusion hybrids had exactly theexpected chromosome composition, while eachof the three aneuploid hybrids had lost twochromosomes of S. etuberosum. Twobackcross progenies were developed, andGISH analysis was applied to analysetransmission of the parental chromosomesinto the sexual generations. BC₁hybrids derived from the crosses of thehexaploid somatic hybrids with tetraploidpotato were pentaploid with thetheoretically expected genomic compositionor with slight deviation from thisexpectation. In the three BC₂ hybridsanalysed by GISH seven to 12 chromosomes ofS. etuberosum were detected in thepredominant S. tuberosum background.No recombinant chromosomes in the hybridswere detected. Genome dosage affects tuberformation in hybrids and their progenies,but has less effect on resistance to potatovirus Y (PVY) in fusion hybrids. Severalgenotypes of the fusion hybrids andBC₁ progeny did not show viralinfection even in the graftingexperiments.     

Nuclear genetic control of variation in simazine tolerance in oilseed brassicas

Summary Brassica napus is a natural allotetraploid derived from the diploid species B. rapa L. (syn. campestris L.) and B. oleracea L. Somatic hybrids synthesized from highly heterozygous lines of these two diploid species were evaluated for fertility. The hybrids were obtained from two fusion experiments which differed in the B. rapa full-sibling parent used as the source of protoplasts. Both B. rapa siblings were lelf-incompatible (SI) yet contained different S-alleles; the B. oleracea species parent was self-compatible (SC). Eight tetraploid hybrids examined had very high female and male fertility; eight hybrids with higher ploidy had low fertility. Hybrids derived from one B. rapa sibling were self-incompatible, whereas those derived from the other B. rapa sibling were fully self-compatible. These data suggest that the different S-alleles of each B. rapa sibling displayed varying penetrance relative to the SC of the B. oleracea parent when combined in B. napus.Abbreviations SC self-compatibility - SI self-incompatibility     

Transfer of resistance to race 2 of Plasmodiophora brassicae from Brassica napus to cabbage (B. Oleracea spp. Capitata). IV. A resistant 18-chromosome B1 plant and its B2 progenies

Summary The somatic karyotype and meiotic chromosome behavior were studied in an 18-chromosome B₁ plant derived from backcrossing a triploid (Brassica napus x B. oleracea ssp. capitata) F₁ hybrid to cabbage. It is considered that cabbage chromosomes no. 1 and no. 7 were substituted by two shorter B. napus chromosomes. Meiotic disturbances were more apparent during the late stages of second division. Seed fertility of this plant was largely restored in the second backcrosses with both cabbage and broccoli. 18-chromosome B₂ plants resistant to race 2 of Plasmodiophora brassicae were recovered among the progenies.Contribution no. J. 725 from the Research Station, Research Branch, Agriculture Canada, St-Jean, Québec J3B 6Z8.     

Resynthesis of rapeseed (Brassica napus L.): A comparison of sexual versus somatic hybridization

Sexual and somatic Brassica napus hybrids produced from the same parental plants were compared. Sexual crosses between a white-flowered, self-compatible broccoli selection (B. oleracea var. italica, cc genome) as the maternal parent and a flowering pak choi accession (B. chinensis, aa genome) yielded one unique spontaneous hybrid and four hybrids through embryo rescue. Thirty-nine somatic hybrids were recovered from a protoplast fusion experiment. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Sexual and somatic hybrids exhibited differences in leaf morphology, flower colour, flowering habit, and organellar inheritance. Sexual hybrids were all fertile amphidiploids (2n = 38, aacc) following spontaneous chromosome doubling. All somatic hybrids had high nuclear DNA contents; most were probably hexaploids (aaaacc or aacccc) from the fusion of three portoplasts. Two initially sterile hexaploid (aaaacc) regenerates eventually set selfed seed after the loss of the putative extra aa genome following regrowth from axillary buds. A bias toward inheritance of B. chinensis chloroplasts was observed with somatic hybrids.     

Disease response of resynthesized Brassica napus L. lines carrying different combinations of resistance to Plasmodiophora brassicae Wor.

Resistance responses of resynthesized Brassica napus lines to infection with Plasmodiophora brassicae were investigated. Lines that were derived from interspecific crosses between clubroot-resistant B. rapa and resistant B. oleracea exhibited very broad and effective resistance in both greenhouse and field tests. When clubroot resistance was introduced into resynthesized lines from the B. oleracea parent only, the plants were mainly susceptible. Interspecific hybrids from the most resistant parental genotypes, i.e. B. campestris ECD-04 and the B. oleracea cultivars ECD-15 or ‘Bohmerwaldkohf’, were used to initiate a B. napus resistance-breeding programme. These artificial rapeseed lines were resistant to isolates that were virulent on all B. napus differential lines and/or parental lines. Preliminary segregation analysis suggests that their resistance is due to at least two dominant and unlinked genes. In some cases progenies from selfed resynthesized plants exhibited resistance reactions that differed from those of the parental hybrid plant; this may have been the result of cytological instability.     

Gene flow from transgenic oilseed Brassica juncea (L.) Czern. into weedy Sinapis arvensis L. (wild mustard)

Gene flow from acetolactate synthase‐resistant (HR) Brassica juncea oilseed canola to related weed, Sinapis arvensis (density 1 plant/m²) was assessed in a 100 m² field plot of HR B. juncea. Two HR F₁ hybrids (H1 and H2) were detected among 109 951 seedlings screened with imazethapyr (hybridization frequency – 1.8 × 10^?5). Hybridity was confirmed using flow cytometry, B. juncea‐specific amplified fragment length polymorphisms (AFLPs) markers, genomic in situ hybridization (GISH) and PCR‐based detection of B. juncea's HR gene. H1 and H2 had 2n = 27 and 2n = 45 chromosomes, corresponding 3x (SrAB) and 5x (SrAABB) genomic structures and reduced male fertility, 3.2 and 16.6% pollen viability, respectively. H1 was self‐incompatible, whereas H2 set seed when selfed (B. juncea trait). Selfed F₂, F₃ and F₄ plants showed HR trait persistence and vigorous growth and high (80–100%) pollen fertility in 22% and 39% of the F₂ and F₃ plants, respectively. No progeny were obtained from F_1, F₂ or F₃ hybrids × S. arvensis backcrosses, suggesting the likelihood of introgression of traits is low to negligible.     

Introgression of Cajanus platycarpus genome into cultivated pigeonpea, C. cajan

Summary Cajanus platycarpus, an incompatible wild species from the tertiary gene pool of pigeonpea (C. cajan (L.) Millspaugh), has many desirable characteristics for the improvement of cultivated varieties. To necessitate such transfers, embryo rescue techniques were used to obtain F₁ hybrids. The F₁ hybrids were treated with colchicine to obtain tetraploid hybrids, that were selfed to obtain F₂, F₃ and F₄ progenies. All of the hybrids and subsequent progenies had an intermediate morphology between the two parents. Backcrossing of the tetraploid hybrids with cultivated pigeonpea was not possible given embryo abortion, with smaller aborted embryos than those obtained in the F₀ parental cross.As a route of introgression, diploid F₁ hybrids were backcrossed with cultivated pigeonpea and BC₁ progeny obtained by in vitro culture of aborting embryos. BC₂ plants were obtained by normal, mature seed germination. Although embryo rescue techniques had to be used to obtain F₁ and BC₁ plants, it was possible to produce BC₂ and subsequent generations through direct mature seed. Every backcross to cultivated pigeonpea increased pollen fertility and the formation of mature seeds.Special project assistant till December, 2003.     

Mapping of Re,a gene conferring the red leaf trait in ornamental kale (Brassica oleracea L. var. acephala)

Variegated leaf colour is an important agronomic trait that affects the market value of ornamental kale (Brassica oleracea L. var. acephala). The red leaf phenotype in kale is due to anthocyanin accumulation. To investigate the pattern of inheritance of this trait, we constructed an F₂ population by crossing ‘Y005‐15’, a double haploid with red leaves, with a white‐leaved double haploid, ‘Y011‐13‐38’, followed by self‐pollination. An F₂ population consisting of 4284 individuals was used to study the inheritance of this trait, which showed that the character was controlled by a dominate gene. All of the 1050 white leaf trait plants in the F₂ were used for mapping and developing markers linked to Re gene. Results showed that Re was mapped to a locus on linkage group C09 of Brassica oleracea, and the locus was mapped between six SSR markers (C9Z1, C9Z16‐1, C9Z90, C9Z94, C9Z96 and C9Z99), with a genetic distance of 6.7, 1.0, 0.3, 2.0, 2.1 and 0.4 cM from Re gene, respectively. These results may facilitate marker‐assisted selection of the red leaf trait in kale breeding as well as map‐based cloning of the red leaf trait gene.