Colchicine-induced heritable variation in cell size and chloroplast number in the leaf cells of inbred ryegrasses (Lolium perenne,L. multiflorum)

Summary A 3 hr period of treatment with a 0.2% aqueous solution of colchicine was given to one week old seedlings of 3 inbred lines of perennial ryegrass (Lolium perenne) and 4 lines of Italian ryegrass (L. multiflorum). The surviving plants developed as mixoploids and were self-pollinated. In the following generation (CT1) the seeds were grown without further treatment, and were screened cytologically at the seedling stage to separate out diploids from tetraploids. Only the diploids were kept, and in this CT1 generation comparisons were then made between controls (2x) and colchicine-treated diploids (C2x) for leaf mesophyll cell size and chloroplast number variation. Heritable differences were found such that the cell plan areas and/or the numbers of chloroplasts per cell were significantly greater in the C2x compared with the 2x treatment in 3 and 5 out of the 7 lines. In each case there was enhancement of chloroplast numbers independently of the variation in cell size.     

Colchicine-induced variation of cell size and chloroplast number in leaf mesophyll of rice

Comparisons were made in the C₁ generation between the untreated controls (2x) and colchicine‐treated diploids (C2x) for variation of leaf mesophyll cell size and chloroplast number. Differences were found in that the cell section area and the number of chloroplasts per cell in all the seven lines investigated were significantly greater in the C2x selections compared with the 2x controls using coded determination. In each case, there was an enhancement of chloroplast number independent of the variation in cell size.     

Utilizing Arachis cardenasii as a source of Cercospora leafspot resistance for peanut improvement

Summary Advanced generation 40-chromosome hybrids between A. hypogaea (2n=4x=40) and a wild diploid species. A. cardenasii were evaluated for early leafspot, Cercospora arachidicola resistance and agronomic potential. The objective of this investigation was to determine if early leafspot resistance derived from a wild species could be incorporated into the A. hypogaea genome. Interspecific hybrid selections were made and then compared in the field and greenhouse to susceptible cultivars and to A. hypogaea lines which are reported to be resistant to early leafspot. Significantly higher levels of resistance were found in five hybrid selections than in cultivated lines based on numbers of lesions per leaf. In a greenhouse study, several hybrid selections also had greatly reduced sporulation from lesions as compared to A. hypogaea. Several mechanisms of resistance are believed to be present. Although hybrid selections had small seeds and low yields as compared to A. hypogaea, a new and valuable source of early leafspot resistance derived from the species A. cardenasii is present.Paper no. 8814 of the journal series of the North Carolina Agricultural Research Service, Raleigh, NC 27695. This research was partially funded by AID-Peanut CRSP grant DAN-4048-G-SS-2065-00.     

AFLP analysis of introgression in coffee cultivars (Coffea arabica L.) derived from a natural interspecific hybrid

S.288 an offspring of a putative spontaneous interspecific hybrid between tetraploid Coffea Arabica (2n = 4x = 44) and diploid C. liberica(2n = 2x = 22) and 17 arabica coffee introgression lines (representing F₂ and F₄) derived from the cross S.288 x Kent arabica were evaluated for introgression of C. liberica genetic material. In all, 36 AFLP primer combinations were used in the analysis. The AFLP profiles of introgressed lines were compared to five accessions each of C. arabica and C. liberica. A total of 137 polymorphic bands were scored among the 29 accessions analysed. The introgressed genotypes exhibited 102 marker bands consisting of 65 additional bands and 37missing bands associated with introgression of C. liberica genetic material. C. liberica accessions of EA group (C. liberica var liberica of Guinean origin) seemed to be the likely progenitor in the origin of natural hybrid. Analysis of genetic relationships in the introgressed lines suggested that introgression was limited to few fragments. Segregation and wide variation in number of marker fragments in the F₂ and F₄progenies were attributed to chromosome recombinations. The differences in the level of introgression between introgressed parent, F₂ and F₄ groups was not pronounced. Therefore the alien genetic material appeared to be fixed and there was no elimination or counter-selection over generations, from introgressed parent to F₄.In C. arabica accessions, only 35 polymorphic bands were seen confirming the low genetic diversity. On the contrary, although representing a small amount of alien genome introgression, the Liberica-introgressed genotypes provided notable genetic diversity. Considering the fact that the diploid species of Coffea constitute a valuable source of genetic diversity, the potential implications of variability generated by Liberica-introgressed genotypes in C. arabica breeding are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Use of Vaccinium octoploids to facilitate 4x-6x gene transfers

The use of an 8x Vaccinium produced through doubling a tetraploid hybrid with colchicine was studied as a means of facilitating gene exchange between V. corymbosum L. and V. ashei Reade. Analysis included meiotic observations and crossability studies of the 8x plant, as well as attempting to develop 6x breeding lines. Meiotic analysis revealed the presence of micronuclei in Telphase II products and polyspory in sporads. In the crossability studies five 4x highbush plants were pollinated with 8x o2 4x pollen. Data gathered included: % fruit set, average weight/fruit, average number of seeds/fruit, average number of seedlings/pollination, and number of 6x seedlings. Octoploid pollinations were significantly lower than 4x pollinations in all parameters. One 6x plant was produced from the 4x-8x crosses but was found to be mitotically unstable, having somatic cells with chromosome numbers ranging from 48 to 168.     

Honeycomb selection for yield in early generations of spring wheat

Summary The effectiveness of the honeycomb selection method for yield in spring wheat (Triticum aestivum L.) was evaluated using progenies from two wheat crosses, Glenlea x NB131 and Glenlea x Era. Honeycomb selection was carried out in the F₂ and F₃ generations, grown at the University of Manitoba in the summers of 1980 and 1981, respectively. In both generations, divergent selection was made for both high and low yield. Plants selected in the F₃ generation were entered in an F₄ yield test in the summer of 1982. Results of the experiment showed that honeycomb selection for yield in the F₂ and F₃ generations was effective in identifying parents of high- and low-yielding lines. F₃ plants from highyielding F₂ selections gave higher yields than those from low-yielding F₂ selections by 11.5% and 13.0% for Glenlea x NB131 and Glenlea x Era crosses, respectively. The F₄ yield test showed that high yielding selections from both crosses significantly outyielded by 8.9% low yielding selections and by 14.4% the unselected composite lines. It is concluded that the honeycomb selection method can be used for early generation selection in spring wheat.     

Partial control of chromosome elimination by temperature in immature embryos of Hordeum vulgare L. x H. bulbosum L.

Summary Embryos derived from Hordeum vulgare L. x H. bulbosum L. were subjected in vivo to a range of temperatures, and the proportions of hybrid plants which retain both parental sets of chromosomes were determined. Elimination of the H. bulbosum genome was significantly increased at temperatures greater than 20°C and resulted in fewer hybrid plants compared with temperatures below 17.5°C. Embryos were also allowed to develop in situ at 15°C and then transferred to 26°C for 8, 16 or 24h during the first 7 days after pollination. A period of 16 h at 26°C (equivalent to at least one complete mitotic cycle) at 2–5 days after pollination was found to be sufficient to increase chromosome elimination significantly above the levels obtained at a constant 15°C. At this stage (2–5 days after pollination at 15°C) the mean embryonic cell number was 2.3–223.     

Expression of barley yellow dwarf virus and Russian wheat aphid resistance genes in and fertility of spring wheat × triticale hybrids and backcross lines

Summary The Barley Yellow Dwarf Virus disease (BYDV) and the Russian wheat aphid (RWA) Diuraphis noxia (Mordvilko) have caused significant losses to wheat and barley in several areas of the world. Important sources of resistance to both BYDV and RWA have been found in Triticale. Different generations of interspecific wheat x Triticale crosses were produced and the progenies were screened for BYDV and RWA tolerance. Plants with equal chromosome numbers showed different levels of fertility. A significant correlation was observed between pollen fertility and seed set in primary florets (r=0.57). In generaL, pollen fertility, seed set and the number of euploid plants (2n=42) increased from one generation to the next. The expression of BYDV tolerance varied from population to population. Additive effects were predominant in F₁ and some backcross populations. A dominant effect of rye tolerance genes was also observed in few populations. A monogenic trait or a quantitative (polygenic) character would not agree with the observed segregation patterns. The heritability of this oligogenic tolerance was quite different between populations and in many populations the tolerance genes were only partially expressed. Some transgressive segregation for tolerance and sensitivity was demonstrated. The genes controlling tolerance to RWA in Triticale lines, Muskox 658 and Nord Kivu were not expressed in advanced lines resistant to BYDV. This indicates that tolerance genes for BYDV and RWA in these lines are located on different chromosomes.     

High frequency regeneration and heritable somaclonal variation in Brassica juncea

Summary Multiple shoot formation in cotyledonary callus of Indian mustard (Brassica juncea cv. Prakash) was induced on modified MS media supplemented with high cytokinin (kinetin or zeatin) and low IAA concentrations. Complete plants were obtained on prolonged incubation of shoots on the same medium. 6-Benzyladenine alone or in combination with IAA or NAA did not support plantlet regeneration. A total of 71 plants were transferred to greenhouse. The seed, however, could be collected from 37 plants only. The seed was sown in the field to evaluate the material for somaclonal variation in R₁ generation. Data were recorded for yield, plant height, number of primary branches, siliqua number, 1,000 seed weight and oil content. Somaclonal lines showed tremendous amount of variation for all the characters studied. A number of plants in this generation showed significantly higher yield and/or other improved agriculturally important characteristics as compared to the control. A line with dwarf plant type was also identified. A number of plants were selected from this generation and carried forward to R₂ generation. Most of these lines bred true in R₂ generation. The material seems to be very promising for future breeding programmes.     

The production of multispecific octoploids from Fragaria species and the cultivated strawberry

Summary A method of incorporating genetic material from five species of Fragaria and the cultivated strawberry into fertile octoploids is described. A synthetic octoploid derived from a hexaploid x diploid hybrid was crossed to octoploid cultivar breeding lines until a BC₂ hybrid was produced. A second synthetic octoploid in which two diploid species and a tetraploid species were combined was crossed to a cultivated strawberry to produce a hybrid breeding clone. The two breeding clones were crossed and 222 seedlings were produced. The seedlings were generally vigorous and fruitful, some having commercial potential.     

Development of interspecific somatic hybrid cell lines in cultivated jute and their early characterization using jute chloroplast RFLP marker

A strong sexual incompatibility barrier that exists between the two cultivated jute species, Corchorus capsularis and Corchorus olitorius, limits the scope for improvement through genetic introgression. Protoplast fusion was carried out to generate interspecific hybrid cell lines. Cotyledonary cell protoplasts of C. capsularis and anthocyaninpigmented hypocotyl protoplasts of C. olitorius were used in the fusion experiments, which appeared to be visually useful in the early selection of the fused products. A chloroplast DNA (cpDNA) marker was developed in jute, which showed species‐specific hybridization patterns with EcoRI‐digested total genomic DNA of C. capsularis and C. olitorius. This cpDNA marker was used in the characterization of the somatic hybrid cell lines at their early stages of growth. Evidence for the presence of both types of cpDNA in the hybrid cell lines was obtained when the total genomic DNA of 4‐ to 7‐month‐old hybrid cell lines was challenged with the chloroplast DNA marker through Southern analysis. It was shown that the early segregation of the parental chloroplasts did not occur in jute, although this is common in other plant species. The hybrid nature of the fused cell lines could also be identified through peroxidase isozyme analysis. Isozyme banding patterns were complex and varied among the hybrid cell lines.     

Development of an appropriate breeding scheme for tolerance to Empoasca kraemeri in common bean

Summary Significant advances in increasing tolerance to the leafhopper Empoasca kraemeri Ross & Moore in common bean have been obtained using a new breeding scheme where yield under leafhopper attack is the principal selection criterion in the evaluation of progenies. However, to further refine this breeding scheme, a study was conducted to determine whether selection for nonprotected yield would be more effective in early versus late generations. Two selection strategies were compared. In Strategy I, early generation selections in the F₂ and F₃ were compared to Strategy II where late generation selections were made in the F₄ and F₅, with the F₂ and F₃ generations advanced using single pod descent and bulk practices, respectively. Yield trials of the F₆ lines from both selection strategies were conducted under nonprotected and insecticide protected treatments. No significant differences were detected between the two selection strategies. However, Strategy II did produce advanced lines with greater nonprotected yields than did Strategy I, with the best F₆ line, in three out of four crosses, coming out of Strategy II. Late generation selection is recommended over early generation selection. Results of the new leafhopper breeding scheme, based on yield, are compared to the old breeding scheme where selections were made using visual selection practices in early generations.     

Heterosis and epistasis in rapeseed estimated from generation means

Summary Heterosis and epistasis in spring oilseed rape (Brassica napus L.) was analysed by comparing generation means for ten agronomic traits. Parents, F₂, F₃ and F₆ generations of four crosses with Swedish and French material were investigated. The F₂ was 11% higher in yield, earlier in flowering time, and slightly later in maturation when compared with the parents. Randomly derived single seed descent lines had an 8% lower yield, were later flowering and maturing than the parents in F₆. This poorer average performance of recombinant lines is explained by the loss of favourable epistatic interactions present in the parents.     

Characterisation of transgenic oilseed rape expressing pea lectin in anthers for improved resistance to pollen beetle

In order to evaluate pea lectin as a resistance factor against the pollen beetle (Meligethes aeneus), transgenic oilseed rape (Brassica napus) has been produced wherein the pea lectin gene expression is driven by a pollen-specific promoter. The aim of the present study was to identify and characterise non-segregating transgenic and non-transgenic lines to be compared in various future tests for benefits and risks associated with such transgenic crop plants. Three doubled haploid (DH) populations (1436, 1440 and 1451) expected to include lectin-producing lines and two DH populations (1443 and 1449) expected to be free of lectin were produced. All five populations originated from different transformation events in the cultivar Westar. The relative amounts of DNA from the marker gene cassette were quantified by conventional as well as real-time PCR analyses and lectin concentrations were estimated by western blot analysis. Two populations with high lectin concentrations, 1436 and 1451, contained higher amounts of the marker DNA and thus more lectin gene copies as compared with 1440 which had lower concentrations of the lectin. As expected all DH lines from 1443 and 1449 were free of lectin. Maximum pea lectin concentration obtained corresponded to 3% of total soluble protein in the anthers. There were significant differences between the populations with respect to bud and flowering stage phenology as well as seed yields, but the differences were not related to their transgenic status. All in all there were 171 lines tested for phenology and transgenic status, out of which 89 with similar phenology were subjected to tests for lectin concentration and seed yield, and 20 of these lines were retested in the next generation. Finally two lines with high lectin concentrations and one with an intermediate level along with two matching non-transgenic lines were selected for future benefit/risk experiments.     

Improved frost tolerance and winter survival in winter barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) by <Emphasis Type="Italic">in vitro</Emphasis> selection of proline overaccumulating lines

Embryogenic calli derived from anther cultures of the two-rowed winter barley cultivar Igri were plated on solid L3 medium containing the proline analogue hydroxyproline (Hyp), 10–20 mmol l^–1. Exposure to Hyp caused severe degeneration of most of the calli. Hyp resistant calli, distinguishable by their lighter colour and higher growth rate, and control calli not exposed to Hyp were plated on L3 regeneration medium. From 22,500 anthers exposed to Hyp 46 Hyp resistant regenerates were obtained, which were transferred to soil. After cultivation for 5–10 weeks at normal growth conditions they were cold hardened at 2 C under short day conditions together with control regenerates. Frost tolerance assays with segments of fully grown leaves of unhardened and cold hardened plants revealed that Hyp resistant regenerants were significantly more frost tolerant than the control regenerants. Improved frost tolerance was found also in the progenies R₁ to R₉, and genotypic segregation in the R₁ generation in a 1:2:1 ratio was indicated. Increased proline content was observed in the R₂ generation and in subsequent generations and was significantly (P 0.001) correlated with increased frost tolerance in the Hyp lines. Comparative studies of R₉ progenies from homozygous R₂ plants with the wild type Igri under field conditions in winter at three locations in Europe as well as crossing experiments confirmed the heritable improvement of frost tolerance and winter survival, respectively, in the Hyp lines. The results support the hypothesis that proline accumulation in cold acclimated winter barley plants is causally related to the acquisition of frost tolerance. Moreover, the described biotechnological procedure may be applicable in breeding programs for improved winter hardiness and possibly also for other stress tolerances.     

Comparative efficiency of honeycomb and conventional pedigree methods of selection for yield and fiber quality in cotton (Gossypium spp.)

The effectiveness of honeycomb pedigree selection (HPS) as compared to conventional pedigree selection (CPS) was studied in one intraspecific (G. hirsutum L. × G. hirsutum L.) cross population (population I) and one interspecific (G. hirsutum L. × G. barbadense L.) cross population (population II). Combined selection for yield and lint quality traits was applied for four cycles at two locations for population I and atone location for population II. Finally, the best F₆ lines derived by each method, together with the unselected population derived by single seed descent (SSD) and three check cultivars, were tested in comparative experiments, separately for each cross, at the same locations. In both populations the analysis of variance indicated significant differences among the groups of the material tested for seed cotton yield, mean boll weight, micronaire reading, staple length, and uniformity ratio. No significant differences were found with respect to plant height, lint percentage, and fiber strength in population I and with respect to lint percentage and fiber strength in population II. In population I, on the basis of mean seed cottonyield and number of superior lines derived by each method as compared to the check varieties and the unselected SSD population, HPS-lines were superior to CPS-lines for seedcotton yield, fiber length and boll weight. HPS-lines, however, did not differ significantly in seedcotton yield from the best check cultivar Eva. Finally, the material derived by CPS was earlier in maturity than the material derived by HPS and SSD. Also in population II, on the basis of mean seedcotton yield and number of superior lines derived by each method as compared to the unselected SSD population, HPS-lines were superior to CPS-lines. No significant differences, however, were identified between the material of the two methods for lint quality traits. It was concluded that in both populations HPS was more effective than CPS in identifying lines with high yielding ability and good lint quality. This superiority of HPS is attributed, at least partially, to its effectiveness in early generation selection. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rate and inheritance of cross-fertilization in faba bean (Vicia faba L.)

Summary To assess the feasibility for synthetic varieties of Vicia faba L., 108 faba bean genotypes were screened for cross-fertilization rate in five field experiments carried out in 1989 and 1990 at Wageningen.Significant genotypic differences were observed for outcrossing rate, which ranged from 1 to 55%. These percentages, however, are thought to be not high enough for a commercially feasible production of synthetic varieties.The inheritance of cross-fertilization rate, determined by testing the F₄ generation of a cross between two inbreds, both with high outcrossing rates, appeared to be of a polygenic nature, as was indicated by the wide variation in, and the continous distribution of, the cross-fertilization rates. Among the F₄ lines tested some had a significantly higher cross-fertilization rate than the parents. However, this difference was not found when these lines were retested, indicating that possibilities for successful selection for higher outcrossing rates might be limited.     

Comparison of selection procedures in breeding for seed yield in segregating sesame populations

Summary Four hundred sesame (Sesamum indicum L.) lines selected by five methods from five crosses were tested for yield and other agronomic characters in F₆ and F₇. More high yielding lines were isolated by the bulk method and in the cross involving two local cultivars when two seasons means and performance in each individual season were considered. The cross involving two exotic cultivars did not produce a single line superior to local recommended variety. The 80 highest yielding lines recorded a 10.8% increase in yield compared to the mean of 400 lines. Thirty five lines were among the top 20% both seasons with a 13.2% increase in yield. Highly significant seasonal and population effects were recorded. There were no significant differences among the methods of selection although the yield differences among lines were highly significant. Interactions of seasons × populations, seasons × methods, populations × methods and seasons × populations × methods were highly significant. The results indicate that simple and less expensive bulk method may be used with equal success as the pedigree, single seed descent or early generation testing methods when selecting for yield in segregating sesame populations. The importance of inclusion of a high yielding, locally adapted cultivar in the crossing programmes for yield breeding of sesame has also been revealed.Abbreviations BM Bulk method - EGT Early generation testing - G × E Genotype × environment - MSS Modified single seed descent - PM Pedigree method - SSD Single seed descent     

叶绿体型转昆虫抗冻蛋白基因烟草的耐寒性

根据已构建的大豆叶绿体表达载体pJY01,设计特异性引物,将昆虫抗冻蛋白基因MpAFP149插入此载体中构成叶绿体表达载体pJY01-MpAFP149,利用基因枪轰击法转化烟草,经壮观霉素筛选获得4株叶绿体型转抗冻蛋白基因烟草株系。PCR和PCR-Southern结果显示外源基因已整合至烟草叶绿体基因组中但同质化水平不高,RT-PCR结果也表明昆虫抗冻蛋白基因已发生了转录。将野生型烟草、叶绿体型转抗冻蛋白基因烟草及核转化T₁代转抗冻蛋白基因烟草(pCAMBIA1302- MpAFP149)于–1℃低温处理3 d,观察耐寒表型及测定相对电导率。结果表明, 叶绿体型转基因烟草的耐寒表型优于野生型烟草,但与核转化的T₁代转抗冻蛋白基因烟草无显著差异。处理3 d时,叶绿体型转基因烟草和T₁代转抗冻蛋白基因烟草的电导率分别为39.2%和38.2%,而野生型烟草已达73.7%。本实验获得的异质化转叶绿体抗冻蛋白基因烟草与转核基因烟草的耐寒力无差异。     

A self-fertile trigeneric hybrid in the Triticeae involving Triticum,Hordeum, and Secale

Summary A self-fertile trigeneric hybrid in the Triticeae involving species from the Hordeum, Triticum and Secale genera has been produced. The trigeneric hybrid was obtained by crossing octoploid triticale (x Triticosecale Wittmack) with octoploid tritordeum (H. chilense × T. aestivum amphiploid). The trigeneric hybrid presented a genome constitution AABBDDRH^ch and 2n=8X=56 chromosomes. The cytogenetical analyses showed no chromosome instability nor homeologous pairing between Hordeum and Secale chromosomes. In the F₂ generation the chromosome number ranged from 42 to 52. Within this range, the plants with smaller numbers of chromosomes were more frequent. A preferential transmission of rye chromosomes could be inferred.