Synthesis of hexaploid (AABBCC) somatic hybrids: a bridging material for transfer of ‘tour’ cytoplasmic male sterility to different Brassica species

Most of the alloplasmic cytoplasmic male sterility (CMS) systems are known to be associated with a number of floral abnormalities that result from nuclear-cytoplasmic incompatibilities. One such system, tour, which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp. While the restorer for this CMS has been reported to be present in B. napus, in B. juncea, where the abnormalities are more pronounced, no restorer has yet been identified. Rectification of these floral abnormalities through mitochondrial recombinations and chloroplast replacement might result in the improvement of this CMS system. As organelle recombinations can possibly be achieved only by somatic cell hybridization, fusion experiments were carried out between hygromycin-resistant B. juncea AABB carrying tour cytoplasm and phosphinotricin-resistant, normal B. oleracea CC to generate AABBCC hexaploid somatic hybrids. The presence of selectable marker genes facilitated the selection of hybrids in large numbers. The resulting hybrids showed wide variation in floral morphology and organelle composition. Regenerants with normal, male-sterile flowers having recombinant tour-or oleracea-type mitochondria and oleracea-type chloroplasts were obtained. Hybrids with male-fertile flowers were also obtained that had recombined tour mitochondria. The AABBCC hexaploid hybrids synthesized in the present study were successfully utilized as a bridging material for transferring variability in the organelle genome simultaneously to all the digenomic Brassica species, and all of these hybrids are now being stabilized through repeated backcrosses to the allopolyploid crop brassicas.     

Unilateral incompatibility as a major cause of skewed segregation in the cross between Lycopersicon esculentum and L. pennellii

Skewed segregations are frequent events in segregating populations derived from different interspecific crosses in tomato. To determine a basis for skewed segregations in the progeny of the cross between Lycopersicon esculentum and L. pennellii, monogenic segregations of 16 isozyme loci were analyzed in an F₂ and two backcross populations of this cross. In the F₂, 9 loci mapping to chromosomes 1, 2, 4, 9, 10 and 12 exhibited skewed segregations and in all cases there was an excess of L. pennellii homozygotes. The genotypic frequencies at all but one locus were at Hardy-Weinberg equilibria. In the backcross populations, all except two loci exhibited normal Mendelian segregations. No post-zygotic selection model could statistically or biologically explain the observed segregation patterns in the F₂ and backcross populations. A pre-zygotic selection model, assuming selective elimination of the male gametophytes during pollen function (i.e., from pollination to karyogamy), could adequately explain the observed segregations in all three populations. The direction of the skewed segregations in the F₂ population was consistent with that expected based on the effects of unilateral incompatibility reactions between the two species. In addition, the chromosomal locations of 5 of the 9 markers that exhibited skewed segregations coincided with the locations of several known compatibility-related genes in tomato. Multigenic unilateral incompatibility reactions between L. esculentum pollen and the stigma or style of L. pennellii (or its hybrid derivatives) are suggested to be the major cause of the skewed segregations in the F₂ progeny of this cross.     

Engineering challenges in high density cell culture systems

High density cell culture systems offer the advantage of production of bio-pharmaceuticals in compact bioreactors with high volumetric production rates; however, these systems are difficult to design and operate. First of all, the cells have to be retained in the bioreactor by physical means during perfusion. The design of the cell retention is the key to performance of high density cell culture systems. Oxygenation and media design are also important for maximizing the cell number. In high density perfusion reactors, variable cell density, and hence the metabolic demand, require constant adjustment of perfusion rates. The use of cell specific perfusion rate (CSPR) control provides a constant environment to the cells resulting in consistent production. On-line measurement of cell density and metabolic activities can be used for the estimation of cell densities and the control of CSPR. Issues related to mass transfer and mixing become more important at high cell densities. Due to the difference in mass transfer coefficients for oxygen and CO₂, a significant accumulation of dissolved CO₂ is experienced with silicone tubing aeration. Also, mixing is observed to decrease at high densities. Base addition, if not properly done, could result in localized cell lysis and poor culture performance. Non-uniform mixing in reactors promotes the heterogeneity of the culture. Cell aggregation results in segregation of the cells within different mixing zones. This paper discusses these issues and makes recommendations for further development of high density cell culture bioreactors.     

Inheritance of black sigatoka disease resistance in plantain-banana (Musa spp.) hybrids

Black sigatoka (Mycosphaerella fijiensis Morelet), an airborne fungal leaf-spot disease, is a major constraint to plantain and banana (Musa spp.) production world-wide. Gaining further knowledge of the genetics of host-plant resistance will enhance the development of resistant cultivars, which is considered to be the most appropriate means to achieve stable production. Genetic analysis was conducted on 101 euploid (2x, 3x and 4x) progenies, obtained from crossing two susceptible triploid plantain cultivars with the resistant wild diploid banana Calcutta 4. Segregating progenies, and a susceptible reference plantain cultivar, were evaluated over 2 consecutive years. Three distinct levels of host response to black sigatoka were defined as follows: susceptible (< 8 leaves without spots), less susceptible (8–10) and partially resistant (> 10). Segregation ratios for resistance at the 2x level fitted a genetic model having one major recessive resistance allele (bs ₁) and two independent alleles with additive effects (bsr ₂ and bsr ₃). A similar model explains the results at the 4x level assuming that the favourable resistance alleles have a dosage effect when four copies of them are present in their respective loci (bs _i ⁴ ). The proposed model was further validated by segregation data of S ₁ progenies. Mechanisms of black sigatoka resistance are discussed in relation to the genetic model.     

A self-fertile trigeneric hybrid,Triticum aestivum ×Agropyron michnoi ×Secale cereale

Trigeneric hybrids between the (Triticum aestivum ×Agropyron michnoi) F₁ (CM, 2n=5x=35; ABDPP) and two winter rye (Secale cereale L., 2n=2x=14; RR) cultivars, Wugong 774 and AR-132, were synthesized. Such trigeneric hybrids could be used to transfer resistance genes for powdery mildew from rye to CM and subsequently to common wheat and to identify (1) the effects of the P genome ofAgropyron on the self-fertility of the hybrids and (2) the differences in genetic background between rye cultivars with marked differences in pollinating habit. The trigeneric hybrids varied widely in morphology and showed a high level of resistance to such diseases as barley yellow dwarf virus (BYDV), stripe rust, leaf rust, stem rust, and powdery mildew. Selfed and many backcross derivatives were obtained from the trigeneric hybrids. The results indicated that rye cvs Wugong 774 and AR132 arose from different gene pools and that the P genome ofAgropyron carries gene(s) responsible for chromosome segregation, leading to functional gamete formation and self-fertility of the hybrids. The F₂ and BC₁ plants could be obtained in two ways — fusion of the unreduced gametes and the assumed apomixis of unreduced female gametes in the trigeneric hybrid plant II-4 — which indicates that this trigeneric hybrid may be a special genetic stock. Chromosome pairing in the trigeneric hybrids and ways of producing wheat/rye and wheat/Agropyron translocations are discussed.     

Ultrastructure of Lycopersicon peruvianum leaf explants and streptomycin-resistant shoots on medium containing streptomycin

The effect of streptomycin on morphogenic explants of Lycopersicon peruvianum Mill. was examined microscopically at both the light and ultrastructural level. Early stages in shoot regeneration from leaf explants were distinguished as meristematic tissue at both levels. Small starch grains were observed in the plastids in this tissue but not in plastids in regenerated shoots. In the presence of streptomycin, adventitious shoot regeneration from sensitive leaf strips was inhibited. Large layered bodies were observed within the plastids of sensitive leaf tissue, suggesting the disruption of thylakoid membrane formation. Streptomycin resistant L. peruvianum lines, as well as a chlorophyll-deficient line, were also examined microscopically. The chloroplasts of newly regenerated streptomycin resistant shoots contained well developed internal membranes and conspicuous starch grains. Cells containing a mixture of resistant and sensitive plastids were not observed. The plastids in chlorophyll-deficient tissue completely lacked thylakoid membranes, although small vesicles and intraplastid bodies were seen within the stroma.Abbreviations NMU N-methyl-N-nitrosourea     

印产毛喉鞘蕊花的引种栽培研究

本文报道印产毛喉鞘蕊花肉质根稳定性状的分离及栽培技术路线．肉质根株提纯到９９．５％;求得了露地最佳效益种植法,最优水平搭配是：３０ｄ、１５株／ｍ２、高垄、覆膜、底肥、整枝、去花穗,产鲜根８７００ｋｇ／ｈｍ２．     

The cortical contraction related to the ooplasmic segregation inCiona intestinalis eggs

Summary The egg cytoplasm of ascidian,Ciona intestinalis, segregates towards both the animal and vegetal poles within a few minutes of fertilization or parthenogetic activation with ionophore A23187. A constriction appears first on the egg surface near the animal pole and then moves to the vegetal pole. Carmine granules and spermatozoa attached to the egg surface move towards the vegetal pole with the movement of the constriction. Microvilli, which are distributed uniformly in unfertilized egg, disappear on the animal side of the constriction and became more dense on the vegetal side of the constriction. Transmission electron microscopy revealed that sub-cortical cytoplasm, containing numerous mitochondria and sub-cortical granules, moves towards the vegetal pole with the movement of the constriction and then concentrates into a cytoplasmic cap at the vegetal pole. An electron-dense layer appears in the cortex of the cap. The ooplasmic segregation and the cortical contraction were inhibited by cytochalasin B and induced by ionophore A23187. These observations suggest that ooplasmic segregation is caused by the cortical contraction which is characterised by a surface constriction and by the formation of an electron-dense layer.     

The inheritance of restriction fragment length polymorphisms in the flax rust Melampsora lini

Summary Random cDNA sequences synthesized from poly A⁺ RNA extracted from germinated urediospores of the flax rust fungus, Melampsora lini, were used as probes to detect restriction fragment length polymorphisms (RFLPs) in three races of M. lini originating from cultivated flax, Linum usitatissimum, and one race originating from Australian native flax, L. marginale. Fourteen out of 22 probes tested detected RFLPs in the three races from cultivated flax while 19 of the probes detected polymorphisms between these three races and the race from L. marginale. The segregation of seven RFLPs was determined in a family of 19 F₂ progeny derived from a cross between two of the rust races. With six of these the inheritance was consistent, in each case, with the segregation of alleles at a single locus. Inheritance of the seventh was unusual and an explanation involving two loci with null alleles at each was proposed. No linkage was detected between any of the RFLP loci and nine unlinked loci specifying avirulence.