Fertility and chromosome stability in Brassica napus resynthesised by protoplast fusion

Summary Fertile somatic hybrids between Brassica campestris and B. oleracea have been produced by protoplast fusion. Fusion products were identified by their intermediate protoplast morphology. Heterokaryons were isolated either with micropipettes using a micromanipulator or by flow sorting. About 2% of the obtained calli differentiated to shoots. Of the shoots obtained from manually selected heterokaryons, 100% were true hybrids as confirmed by isozyme analysis while 87% of the flow sorted ones showed a hybrid pattern. Ploidy level of the hybrid plants was determined by chromosome counting and relative DNA-content analysis. The sum of the chromosome number (38) from the two fusion partners were found in 30% of the hybrids; 9% had fewer and 61% had more chromosomes. Pollen viability and seed set varied with ploidy level. Compared to natural B. napus, a pollen viability of 52%–93% and a fertility of 1%–40% was found for the somatic hybrids with normal chromosome number. Restriction enzyme analysis of chloroplast-DNA showed that either B. campestris or B. oleracea chloroplasts were present in the somatic hybrid plants. Of 11 hybrid plants 5 had the campestris and 6 had the oleracea type (11 ratio).     

Evidence for spontaneous diploid androgenesis in Brassica napus L.

Summary Seeds of androgenetic origin were obtained among the F₁ progenies of two crosses between resynthesized and cultivated forms of Brassica napus. The high-erucic, white-flowered, resynthesized line No7076 acted as the female, and the zero-erucic, yellow-flowered, cultivars Topas and Puma, as males. No androgenetic seeds were obtained in the reciprocal crosses. Resynthesized rape could thus be of potential use for the production of androgenetic plants. Of special interest is the high frequency (21%) of spontaneous androgenesis observed in one of the two crosses. One plant, determined from erucic acid content and flower colour analysis as androgenetic, had a diploid chromosome number. Further knowledge about the genetic control of spontaneous androgenesis in the present material and the origin of the cytoplasm in androgenetic plants are required in order to exploit this phenomenon in practical plant breeding.     

Studies of protoplast culture and plant regeneration from commercial and rapid-cyclingBrassica species

Protoplasts were isolated from aseptic shoot cultures of commercial cultivars ofBrassica napus, B. oleracea andB. campestris, and from the six rapid-cycling brassica species. Of the rapid-cycling species, onlyB. napus responded well to the culture conditions used; 2% of protoplasts formed calli and up to 5% of calli regenerated shoots. Regeneration was also achieved from commercial cultivars ofB. napus andB. oleracea. For these two species the plating density, time of dilution with fresh medium and the composition of the shoot-inducing medium were all found to have an important influence on the efficiency of plant regeneration. Both responded better to maltose than to sucrose-based media. Under the optimum conditionsB. napus showed a plating efficiency of 7.8% and shooting efficiency of 17%; forB. oleracea the figures were 2% and 56%, respectively.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Transfer of resistance to the beet cyst nematode (Heterodera Schachtii Schm.) from Sinapis alba L. (white mustard) to the Brassica napus L. gene pool by means of sexual and somatic hybridization

Summary Sexual and somatic hybrid plants have been produced between Sinapis alba L. (white mustard) and Brassica napus L. (oil-seed rape), with the aim to transfer resistance to the beet cyst nematode Heterodera schachtii Schm. (BCN) from white mustard into the oil-seed rape gene pool. Only crosses between diploid accessions of S. alba (2n = 24, S_a1S_a1) as the pistillate parent and several B. napus accessions (2n = 38, AACC) yielded hybrid plants with 31 chromosomes. Crosses between tetraploid accessions of S. alba (2n = 48, S_a1S_a1S_a1S_a1) and B. napus were unsuccessful. Somatic hybrid plants were also obtained between a diploid accession of S. alba and B. napus. These hybrids were mitotically unstable, the number of chromosomes ranging from 56 to more than 90. Analysis of total DNA using a pea rDNA probe confirmed the hybrid nature of the sexual hybrids, whereas for the somatic hybrids a pattern identical to that of B. napus was obtained. Using chloroplast (cp) and mitochondrial (mt) DNA sequences, we found that all of the sexual F₁ hybrids and somatic hybrids contained cpDNA and mtDNA of the S. alba parent. No recombinant mtDNA or cpDNA pattern was observed. Three BC₁ plants were obtained when sexual hybrids were back-crossed with B. napus. Backcrossing of somatic hybrids with B. napus was not successful. Three sexual hybrids and one BC₁ plant, the latter obtained from a cross between a sexual hybrid and B. napus, were found to show a high level of BCN resistance. The level of BCN resistance of the somatic hybrids was in general high, but varied between cuttings from the same plant. Results from cytological studies of chromosome association at meiotic metaphase I in the sexual hybrids suggest partial homology between chromosomes of the AC and S_a1 genomes and thus their potential for gene exchange.     

Genetics of self incompatibility in diploid Ageratum houstonianum mill

Summary Diallels and backcrosses among self-incompatible (SI) clones and progeny of Ageratum houstonianum Mill. could be organized into intra-incompatible classes. Four of 5 progenies segregated in expected ratios of S genotypes. Ageratum expressed a one-locus incompatibility system of the sporophytic type with a linear dominance series of multiple alleles and complete allelic dominance in both pollen and stigma. In the second part of the study, a high percentage of self-seed set was observed during the first flowering of a progeny from a pseudo-self compatible (PSC) seed source. Six progenies were derived from the PSC seed source. Five of the 6 segregated PSC SI plants, 4 of which fit a 3 1 ratio of PSC SI plants. All plants of the sixth progeny were SI. Two F₁ progenies with the same PSC pollen parent produced significantly different segregations of PSC SI plants. It appeared that PSC acted as a major gene when the most recessive S allele was also present, but PSC was not expressed when the most dominant S allele was present. Clones propagated from PSC plants were SI and cross incompatible with a related S-allele tester. Thus, PSC was transient in that it was apparent in seed-propagated plants but not in plants clonally propagated from the PSC individuals.Scientific Journal Series Paper Number 12,299 of the Minnesota Agricultural Experiment Station     

Linkage arrangement of restriction fragment length polymorphism loci in Brassica oleracea

Summary A detailed genetic linkage map of Brassica oleracea was constructed based on the segregation of 258 restriction fragment length polymorphism loci in a broccoli × cabbage F₂ population. The genetic markers defined nine linkage groups, covering 820 recombination units. A majority of the informative genomic DNA probes hybridized to more than two restriction fragments in the F₂ population. Duplicate sequences having restriction fragment length polymorphism were generally found to be unlinked for any given probe. Many of these duplicated loci were clustered non-randomly on certain pairs of linkage groups, and conservation of the relative linkage arrangement of the loci between linkage groups was observed. While these data support previous cytological evidence for the existence of duplicated regions and the evolution of B. oleracea from a lower chromosome number progenitor, no evidence was provided for the current existence of blocks of homoeology spanning entire pairs of linkage groups. The arrangement of the analyzed duplicated loci suggests that a fairly high degree of genetic rearrangement has occurred in the evolution of B. oleracea. Several probes used in this study were useful in detecting rearrangements between the B. oleracea accessions used as parents, indicating that genetic rearrangements have occurred in the relatively recent evolution of this species.     

Expression of the C3-C4 intermediate character in somatic hybrids between Brassica napus and the C3-C4 species Moricandia arvensis

The wild crucifer Moricandia arvensis is a potential source of alien genes for the genetic improvement of related Brassica crops. In particular M. arvensis has a C₃-C₄ intermediate photosynthetic mechanism which results in enhanced recapture of photorespired CO₂ and may increase plant water-use efficiency. In order to transfer this trait into Brassica napus, somatic hybridisations were made between leaf mesophyll protoplasts from cultured M. arvensis shoot tips and hypocotyl protoplasts from three Brassica napus cultivars, Ariana, Cobra and Westar. A total of 23 plants were recovered from fusion experiments and established in the greenhouse. A wide range of chromosome numbers were observed among the regenerated plants, including some apparent mixoploids. Thirteen of the regenerated plants were identified as nuclear hybrids between B. napus and M. arvensis on the basis of isozyme analysis. The phenotypes of these hybrids were typically rather B. napus-like, but much variability was observed, including variation in flower colour, leaf shape and colour, leaf waxiness, fertility and plant vigour. CO₂ compensation point measurements on the regenerated plants demonstrated that 3 of the hybrids express the M. arvensis C₃-C₄ intermediate character at the physiological level. Semi-thin sections through leaf tissues of these 3 plants revealed the presence of a Kranz-like leaf anatomy characteristic of M. arvensis but not found in B. napus. This is the first report of the expression of this potentially important agronomic trait, transferred from Moricandia, in M. arvensis x B. napus hybrids.     

The genetics of adult-plant blackleg (Leptosphaeria maculans) resistance from Brassica juncea in B. napus

The genetic control of adult-plant blackleg (Leptosphaeria maculans) resistance in a Brassica napus line (579NO48-109-DG-1589), designated R13 possessing Brassica juncea-like resistance (JR), was elucidated by the analysis of segregation ratios in F₂ and F₃ populations from a cross between R13 and the highly blackleg-susceptible B. napus cultivar Tower. The F₂ segregration ratios were bimodal, demonstrating that blackleg resistance in R13 was controlled by major genes. Analysis of the segregation ratios for 13 F₃ families indicated that blackleg resistance in these families was controlled by three nuclear genes, which exhibited a complex interaction. Randomly sampled plants of F₃ progeny all had the normal diploid somatic chromosome number for B. napus. The similarities between the action of the three genes found in this study with those controlling blackleg resistance in B. juncea is discussed.     

Transfer of resistance to Xanthomonas campestris pv campestris into Brassica oleracea L. by protoplast fusion

Black rot caused by the bacterium Xanthomonas campestris pv campestris is one of the most serious diseases of Brassica oleracea. Since sources of resistance to the disease within B. oleracea are insufficient and control means are limited, the development of resistant breeding lines is extremely desirable. Certain lines of B. napus contain very high resistance controlled by a dominant gene, but crossing the two species sexually is very difficult. Therefore, somatic hybrids were produced by protoplast fusion between rapid cycling B. oleracea and a B. napus line highly resistant to X. campestris pv campestris. Hybrid identity was confirmed by morphological studies, flow cytometric estimation of nuclear DNA content, and analysis of random amplified polymorphic DNA (RAPD). Inoculations with the pathogen identified four somatic hybrids with high resistance. The resistant hybrid plants were fertile and set seed when selfed or crossed reciprocally to the bridge line 15 (Quazi 1988). Direct crosses to B. oleracea were unsuccessful, but embryo rescue facilitated the production of a first-backcross generation. The BC₁ plants were resistant to the pathogen. Progeny from the crosses to line 15 were all susceptible. Embryo rescue techniques were not obligatory for the development of a second-backcross generation, and several resistant BC₂ plants were obtained.     

Inheritance of heat-stable resistance to Meloidogyne incognita in Lycopersicon peruvianum and its relationship to the Mi gene

Summary The inheritance of heat-stable resistance to the root-knot nematode, Meloidogyne incognita (Kofoid and White) Chitwood, was studied in crosses between different accessions and clones of Lycopersicon peruvianum L. F₁, F₂ and BC₁ generations were evaluated for their index of resistance based on numbers of eggs and infective second-stage juveniles (J₂) per gram of root, and the segregation ratios were determined in experiments carried out at constant soil temperatures of 25 °C and 30 °C. L. peruvianum P.I. 270435 clones 3 MH and 2R2 and P.I. 126443 clone 1 MH, all heatstable resistant, were crossed with L. peruvianum P.I. 126440 clone 9 MH, which is susceptible at both 25 °C and 30 °C. All F₁ progeny were resistant at 25 °C and 30 °C; F₂ and BC₁ generations at 25 °C gave resistant: susceptible (RS) ratios of 151 and 31, respectively, which suggests that resistance is conditioned by two independently assorting genes. However, at 30 °C, RS ratios of 31 and 11 were observed for the F₂ and BC₁ generations, respectively. These results indicate that heat-stable resistance is conferred by a single dominant gene expressed at 30 °C, while the second resistance gene is heat unstable and not expressed at 30 °C. P.I. 270435 clones 2R2 and 3 MH and P.I. 126443 clone 1 MH were crossed with P.I. 128657 clone 3 R4 (source of gene Mi), which is resistant at 25 °C but susceptible at 30 °C. All of the F₁ progeny were resistant at 25 °C and 30 °C.TC₁ progeny of 270435-2 R2 x 128657-3 R4, 270435-3 MH x 128657-3 R4 and 126443-1 MH x 128657-3 R4 crossed with susceptible 126440-9 MH were all resistant at 25 °C and segregated in a 11 ratio at 30 °C. These results also suggest that the heat-stable resistance is monogenic and that it is non-allelic to gene Mi. The non-segregation of TC₁ progenies at 25 °C, suggests that the heat-unstable resistance factor in L. peruvianum P.I. 270435 clones 2 R2 and 3 MH and in P.I. 126443 clone 1 MH is allelic to or the same as gene Mi. We propose the symbol Mi-2 for the gene in P.I. 270435 that confers heat-stable resistance to M. incognita.     

Early generation intermating for yield improvement in groundnut (Arachis hypogaea L.)

Summary Seeds of 4 crosses of groundnut (Arachis hypogaea L.), Robut 33-1 x Chico, Robut 33-1 x NC Ac 17090, Robut 33-1 x PI 298115 and MK 374 x GAUG 1, were irradiated with 30 kR. In the F₁, some branches of each plant were intermated with other plants at random and others selfed in each cross to produce S₂ and F₂ seeds. They were evaluated for pod yield, shelling percentage and 100-kernel weight. The frequency of plants superior to F[in1] was much higher in S₂ than in F₂, which was, in general, true for the values of yield and its components. The S₂ and F₂ were advanced to third generation by selfing. The families descending from S₂ showed clear superiority over those from F₂. The reason for such superiority was suggested to be the recombination of genes from the upper and lower ends of the genotypic distribution under intermating.     

Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs)

Summary Preliminary analysis using nuclear RFLPs provided evidence that subspecies within Brassica rapa originated from two different centers. One center is in Europe, represented by turnip and turnip rape from which the oilseed sarson was derived. A second center is in South China containing a variety of Chinese vegetables of which pak choi and narinosa seem to be the most ancient forms. Based on RFLP data, the accessions of B. oleracea examined could be divided into three distinct groups, represented by thousand head kale, broccoli and cabbage. Thousand head kale and Chinese kale appear to be the primitive types. Observations of parallel variation among subspecies of both species are discussed.     

Molecular cloning of a cDNA fromBrassica napus L. for a homologue of acyl-CoA-binding protein

A cDNA encoding an acyl-CoA-binding protein (ACBP) homologue has been cloned from a gt11 library made from mRNA isolated from developing seeds of oilseed rape (Brassica napus L.). The derived amino acid sequence reveals a protein 92 amino acids in length which is highly conserved when compared with ACBP sequences from yeast, cow, man and fruit fly. Southern blot analysis ofBrassica napus genomic DNA revealed the presence of 6 genes, 3 derived from theBrassica rapa parent and 3 fromBrassica oleracea. Northern blot analysis showed that ACBP genes are expressed strongly in developing embryo, flowers and cotyledons of seedlings and to a lesser extent in leaves and roots.     

Molecular marker analysis of Helianthus annuus L. 2. Construction of an RFLP linkage map for cultivated sunflower

A detailed linkage map of Helianthus annuus was constructed based on segregation at 234 RFLP loci, detected by 213 probes, in an F₂ population of 289 individuals (derived from a cross between the inbred lines HA89 and ZENB8). The genetic markers covered 1380 centiMorgans (cM) of the sunflower genome and were aranged in 17 linkage groups, corresponding to the haploid number of chromosomes in this species. One locus was found to be unlinked. Although the average interval size was 5.9 cM, there were a number of regions larger than 20 cM that were devoid of markers. Genotypic classes at 23 loci deviated significantly from the expected ratios (121 or 31), all showing a reduction in the ZENB8 homozygous class. The majority of these loci were found to map to four regions on linkage groups G, L and P.     

Hybridization between Triticum aestivum L. and Agropyron michnoi Roshev.

Summary Intergeneric hybrids between Triticum aestivum cv Chinese Spring (2n=6x=42, AABBDD) and Agropyron michnoi Roshev. (2n=4x=28, PPPP) were obtained by embryo culture. Their spike characteristics were similar to those of common wheat but, unlike their parents, they were long-awned. The average meiotic chromosome pairing at MI of F₁ hybrids was: 6.39 I +3.75 rodII+8.64 ringII+0.81 III+0.30 IV+0.04 V, the bivalent and multivalent formation of which was much higher than expected from the genomic formulae. It is especially worthwhile to note that the F₁ hybrids were self-fertile, self set being 0.15%, and seeds were easily obtained from the backcross of f₁ plants with hexaploid and tetraploid wheats; here the seed set was more than 20.0%. The polyploid taxa and the position of A. Michnoi in Agropyron are discussed.     

Interspecific hybridization of cultivated rice, Oryza sativa L. with the wild rice, O. minuta Presl.

Crosses were made between four varieties (Mahsuri, Setanjung, MR84 and MR103) of Oryza sativa L. (2n=24, AA) and one accession of O. minuta (2n= 8, BBCC). The seed set obtained ranged between 9.5% and 25.1% depending on the rice variety used. By rescuing 14-day-old embryos and culturing them on 25%-strength MS medium we obtained a total of 414 F₁ hybrids. The F₁s were vigorous, tillered profusely, were perennial and male-sterile. The hybrids were triploid (ABC) with 36 chromosomes and showed irregular meiosis. The average frequency and range of chromosome associations at metaphase I or early anaphase I pollen mother cells of F₁ plants were 29.31(16–36) Is +3.32(0–10) IIs+0.016(0–1) IIIs+0.002(0–1) IVs. Upon backcrossing the original triploid hybrids and colchicine-treated hybrids to their respective recurrent parents, and further embryo rescue, 17 backcross-1 (BC₁) plants were obtained. Of all the crosses using MR84, no BC₁ plant was obtained even after pollinating 13 894 spikelets of the triploid hybrid. The BC₁s were similar in appearence to the F₁s and were male-sterile, their chromosome number ranged from 44 to 48. By backcrossing these BC₁s and nurturing them through embryo rescue, we obtained 32 BC₂ plants. Of these, however, only 18 plants grew vigorously. One of these plants has 24 chromosomes and the other 17 have chromosome numbers ranging between 30 and 37. The 24-chromosome plant was morphologically similar to the O. sativa parent and was partially fertile with a pollen and spikelet fertility of 58.8% and 12.5% respectively. All of the F₁ and BC₁ plants were found to be resistant to five Malaysian isolates (XO66, XO99, XO100, XO257 and XO319) of Xanthomonas campestris pv oryzae. Amongst the BC₂s, the reaction varied from resistant to moderately susceptible. The 24-chromosome BC₂ plant was resistant to the four isolates and moderately resistant to isolate XO100 to which the O. sativa parent was susceptible.Part of PhD thesis submitted by first author to Universiti Kebangsaan Malaysia, Bangi     

RFLP mapping of the sugary enhancer1 gene in maize

RFLP marker data from an F₂₃ population derived from a cross between a sugary1 (su1) and a sugary enhancer1 (su1, sel) inbred were used to construct a genetic linkage map of maize. This map includes 93 segregating marker loci distributed throughout the maize genome, providing a saturated linkage map that is suitable for linkage analysis with quantitative trait loci (QTL). This population, which has been immortalized in the form of sibbed F₂₃ families, was derived from each of the 214 F₂ plants and along with probe data are available to the scientific community. QTL analysis for kernel sucrose (the primary form of sugar) concentration at 20 days after pollination (DAP) uncovered the segregation of seven major QTL influencing sucrose concentration; a locus linked to umc36a described the greatest proportion of the variation (24.7%). Since maltose concentration has previously been reported to be associated with the se1 phenotype, an analysis of probe associations with maltose concentration at 40 DAP was also conducted. The highly significant association of umc36a with maltose and sucrose concentrations provided evidence that this probe is linked to se1. Phenotypic evaluation for the se1 genotype in each F₂₃ family enabled us to map the gene 12.1 cM distal to umc36a. In contrast to previous work where se1 was reported to be located on chromosome four, our data strongly suggest that the sugary enhancer1 locus maps on the the distal portion of the long arm of chromosome 2 in the maize genome.     

Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene

Summary Genetically transformed plants of Brassica napus L. (oilseed rape) were obtained from hypocotyl expiants using Agrobacterium tumefaciens vectors. Hypocotyl explants were inoculated with disarmed or oncogenic A. tumefaciens strains, EHA101 and A281, and then cultured on media containing kanamycin. The A. tumefaciens strains harbored a binary vector, which contained a neomycin phosphotransferase II (NPTII) gene driven by the 35S promoter of cauliflower mosaic virus and an engineered napin (seed storage protein) gene with its own promoter (300 nucleotides 5 to the start of translation). Transformation of B. napus plants was confirmed by detection of NPT II enzyme activity, Southern blot analysis and inheritance of the kanamycin-resistance trait (NPT II gene) in the progeny. Expression of the engineered napin gene in embryos but not in leaves of transgenic plants was observed by Northern analysis. These data demonstrate that morphologically normal, fertile transgenic B. napus plants can be obtained using Agrobacterium as a gene vector and that developmentally regulated expression of reintroduced genes can be achieved.     

Callus initiation and regeneration capacities in Brassica species

In order to know the genetic differences of de- and redifferentiation capacities, seven Brassica species (B. campestris, B. nigra, B. oleracea, B. hirta, B. carinata, B. juncea and B. napus) were cultured in vitro, and their response to the medium supplemented with various combinations of auxin and cytokinin hormones was compared. Important factors for callus initiation were shown to be auxin and species. Calli were induced most frequently in Murashige and Skoog (MS) medium with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), whereas -naphthaleneacetic acid (NAA) induced preferentially roots at a concentration of 2 to 5 mg/l. Callus-, root- and shoot-forming capacities from explanted cotyledon tissues were significantly different among the seven Brassica species. Calli derived from cotyledons and hypocotyls of seven species were transferred to MS media with 20g/l sucrose, 0 to 0.1 mg/l NAA and 0 to 4 mg/l kinetin to compare their regeneration capacities. Among the seven species tested, B. napus (2n=4x=38, genome AACC) had the highest shoot forming capacity (20.0%). Other amphiploid species, B. carinata (2n=4x=34, BBCC) and B. juncea (2n=4x=36, AABB) formed shoots at low frequencies (2.8% and 1.2%, respectively). A diploid species, B. oleracea (2n=2x 18, CC) also showed high shoot formation (10.2% on average). This result suggests that the gene(s) controlling shoot formation may be localized in the C genome. Differences were also found among varieties and cultivars within a species. One of the cultivars, Siberian kale (B. oleracea var. acephala) gave about 50% shoot formation. This kale was shown cytologically to be an autotetraploid (2n=4x=36, CCCC), thus probably possessing a double set of the shoot-forming gene(s).     

Genetic variation within and among different cultivars and landraces of Brassica campestris L. and B. oleracea L. based on isozymes

Genetic variation based on isozymes was studied in 43 landraces and cultivars of Brassica campestris from China, 4 cultivars of B. campestris from Sweden and 1 from India, and 5 cultivars of B. oleracea from Sweden and 1 from China (B. alboglabra). A total of 17 isozyme loci was studied, 10 of these were polymorphic in B. campestris and 6 were polymorphic in B. oleracea. The level of heterozygosity seemed to be reduced in the Swedish cultivars compared to the Chinese landraces and cultivars of B. campestris. The level of heterozygosity in B. oleracea was even lower than that in the Swedish cultivars of B. campestris. A phylogeny of the cultivars and landraces of B. campestris showed that the B. campestris var yellow sarson cultivar, originating from India, deviated significantly from the other cultivars of B. campestris. A phylogeny of the cultivars of B. oleracea confirmed the expectations that the cultivar B. alboglabra was not closely related to the cultivated forms of B. oleracea.