Efficient production of transgenic plants by Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz)

An efficient and reproducible method was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. LBA4404(pTOK233), containing the nptII, hph and gus marker genes, was used in the experiments. Chemical selection by means of kanamycin was used to establish 1037antibiotic resistant callus lines, of which 526 showed GUS expression. Of the 241 callus lines that were transferred to maturation medium 219formed somatic embryos. Thirty-seven of the 38 lines that were transferred to germination medium produced plants. GUS-positive plants could be obtained from 31 lines; in 14 of those lines 100% of the produced plants were GUS-positive, the remaining 17 lines yielded GUS-positive plants at an average of 72%. The transgenic nature of these plants was confirmed by Southern blot analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Agronomic and morphological characterization of Agrobacterium-transformed Bt rice plants

The agronomic and morphological characteristics of Agrobacterium-transformed rice plants carrying the synthetic cry1Ab or cry1Ac gene were investigated. Tremendous variations in plant height, seed fertility, grain size and other traits were seen in 80 T₁ lines, derived from 80 T₀ plants of 9 rice varieties. On average, about 33% T₁ lines had either morphological or agronomic variant plants. Most of the variations in T₁ plants had no significant correlation with transgene insertion and were proved heritable to their progenies. Genetic analysis in T₃ or T₄ generations showed some simple mutations such as chlorophyll deficiency and stunted plants were independent of transgene insertion and seemed to be controlled by a pair of single genes. However, in two independent transgenic progenies of Xiushui 11, all plants homozygous for transgenes showed dwarfism while all hemizygous and null segregants had normal plant heights. Two advanced homozygous Bt lines, KMD1 and KMD2, were developed from these two progenies. Comparison of the agronomic traits of KMD1 and KMD2 with their parent displayed marked differences among them in terms of seedling growth, tillering ability, yield components and yield potential. The genetic variation observed was generally not linked to the transgene locus and was ascribed to somaclonal variation, but other causes might also exist in particular cases. The results are discussed in the context of choosing appropriate transformation methodology for rice breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic variation of a safflower germplasm collection grown as a winter crop in southern Spain

Summary A germplasm collection containing 23 accessions of safflower (Carthamus tinctorius L.) was sown in 1993/94 as a winter crop in southern Spain to perform a comparative evaluation. Morphological, physiological and chemical characters were evaluated. Considerable diversity was found in the group. Only one Mexican accession was dwarf (40 cm tall). Most accessions were spiny but there were two spineless Iranian accessions and others with few spines. Variations were also evident for head and seed size. Seed yield per plant varied from 2.5 to 103.5 g and it was correlated with the number of heads, number of branches and plant height. Cultivars with oil content over 35% were identified. Most accessions were of the high linoleic type but there were two with high or intermediate levels of oleic acid, corresponding to the cultivars Ole and Oleic leed. Sowing in November in southern Spain has produced a long growing period (180–240 days) but earliness is common in Indian accessions. A Chinese phenotype with a prolonged rosette stage could be better adapted for cold tolerance. Red flowered spineless cultivars were present in the collection and will be preferred for ornamental or dye purposes.     

Qualitative resistance to powdery mildew in hybrid sweet peas

Lathyrus odoratus L. × Lathyrus belinensis L. hybrids were produced using L. belinensis as the pollen parent, with fertile seed produced by the L. odoratus parent. The F1 hybrid plants were completely self-sterile, but produced viable seeds when backcrossed to L. odoratus. The plants produced by backcrossing resembled L. odoratus, the flower colour being purple/magenta, and were self-fertile. Both hybrid plants and those produced by back crossing to L. odoratus were resistant to Erysiphe pisi DC that causes powdery mildew in sweet peas. Continued backcrossing resulted in hybrid plants, that closely resembled the L. odoratus parent, but segregated for complete resistance/susceptibility to E. pisi,with a ratio of 2.46:1 resistant to sensitive plants. This suggests the presence of a single dominant gene that confers resistance. When resistant and sensitive plants were inoculated with E. pisi and their leaf surfaces examined,using a Scanning Electron Microscope, it was found that although spores germinated on the leaves of both resistant and sensitive plants, spores present on resistant plants collapsed shortly after germination. Possible reasons for this are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

The development of Raparadish (x Brassicoraphanus, 2n=38), a new crop in agriculture

Summary Raparadish, x Brassicoraphanus, the amphidiploid hybrid between Brassica rapa (syn. B.campestris) and Raphanus sativus (fodder radish) was made by Dolstra (1982). Primary hybrid plants grew vigorously, suggesting that the amphidiploid AARR might be useful as a fodder crop. Three populations of this new material were studied, with special attention to improvement of fertility and resistance to beet cyst nematode (Heterodera schachtii), whilst preserving genetic variability. For lack of progress one of the populations was abandoned after the fourth generation. The other two populations were observed through nine or ten generations. Apart from the last two generations mass selection for seed set was carried out on the basis of single plants. This led to a considerable increase in average seed production, without losing a wide variation for this trait. Thus more progress is being expected. Five cycles of mass selection for resistance to beet cyst nematodes led to a considerable increase of the level of resistance of both populations. The prospects of this new agricultural crop are discussed.     

Pollen production in soybeans with respect to genotype,environment, and stamen position

Summary Our objective was to determine the average numbers of pollen grains from fertile plants (Ms ₁) and the average numbers of coenocytic microspores from genetic male sterile plants (ms ₁ ms ₁) in soybeans, Glycine max L. Merr. Comparisons were made between the average numbers of pollen grains and the average numbers of coenocytic microspores with respect to environment where plants were grown and to stamen position in the flower. Five male sterile lines were used. They included the North Carolina ms ₁ mutant, the cultivar Hark with the ms ₁ gene, and mutants identified as the Urbana, Tonica, and Ames male steriles. Three environments used were the Agronomy Farm, University of Georgia, Athens, Georgia; the Agronomy and Agricultural Engineering Research Center, Iowa State University, Ames, Iowa; and the Agronomy Greenhouse, Iowa State University, Ames, Iowa.Pollen production from fertile plants varied from 374 to 760 pollen grains per anther among genetic lines and environments. This variation may be an important consideration in selecting a male parent to use as a pollinator for hybrid seed production.Among fertile plants, the average numbers of pollen grains per anther of the separate stamen and and of the lower whorl of stamens were significantly different only in greenhouse-grown plants. Among male sterile plants, the average numbers of coenocytic microspores per anther of the separate stamen and of the lower whorl of stamens were significantly different in three genotype x environment combinations. These three exceptions did not conform to any genetic or environmental pattern. Deviations from the expected ratio of 4 pollen grains from fertile plants: 1 coenocytic microspore from sterile plants were attributed to initial differences in the average number of microspore mother cells between the two genotypes.Joint contribution: Agricultural Research Service, USDA, North Central Region, and Journal Paper No. J-8910 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011; Project 2107.     

Flow cytometric identification of sexually derived polyploids in hop (Humulus lupulus L.) and their use in hop breeding

The New Zealand hop breeding programme is based solely on the development of seedless triploid cultivars. This relies on the use of tetraploid parents. While a sexually derived tetraploid parents have been used successfully, sexually derived tetraploids offer a useful alternative. They may have a higher level of heterozygosity and are easier to obtain. Methods for the identification of tetraploids from seedling populations by flow cytometry are described. Two studies were conducted; one on field-grown plants, the other on plants grown in a glasshouse. Approximately 15%of seedlings in the two studies were identified as tetraploids (2n = 4x = 40). Between 70 and 80% were identified as either diploid (2n =2x = 20) or triploid (2n = 3x = 30). Remaining plants were haploid, pentaploid or probably aneuploid. Within triploid and tetraploid populations from 40to 50% of seedlings were female, 30 to40% were monoecious, with the remainder being males and non-flowering plants. Sexually derived tetraploid parents have been used successfully in the breeding programme, from which several promising triploid selections have subsequently been made. This revised version was published online in July 2006 with corrections to the Cover Date.     

Yield and fruit quality losses caused by ToMV in pepino (Solanum muricatum L.) and search for sources of resistance

Tomato mosaic virus (ToMV) is a limiting factor for the success of pepino (Solarium muricatum) as a new crop. The effects of ToMV infection on total and marketable yield, fruit weight, length/width ratio and soluble solids content (SSC) have been studied in two commercial clones (`Sweet Long' and `Sweet Round'). ToMV infection depressed total yield in infected plants of `Sweet Long' (43.1%), while it had no effect on this trait in `Sweet Round' . Marketable yield was dramatically reduced by ToMV infection in both clones, 94% in `Sweet Long' and 100% in `Sweet Round'. Infected plants of clone `Sweet Long' had a lower weight than healthy plants. Although no differences in fruit weight were detected in `Sweet Round' between ToMV infected and healthy plants, many fruits from infected plants showed deformities. Changes in fruit length/width ratio and SSC as a result of ToMV infection were not relevant, but fruit quality was lower in infected fruits, most of which had corky-like flesh. Forty-two clones from cultivated (S. muricatum), wild (S. caripense and S. tabanoense) and interspecific hybrids were tested for ToMV resistance. All but seven clones (four from S. muricatum and three from interspecific hybrids S. muricatum × S. caripense) were susceptible. Non-susceptible clones showed variable degrees of resistance and developed hypersensitive local lesions. Among these clones the most promising as sources of variation for resistance to ToMV are those belonging to the cultivated species. Although no immunity was found, plants from these clones remained asymptomatic and absorbance values resulting from the DAS-ELISA tests in these plants were always lower than those of the susceptible control (cv. `Sweet Round'). These sources of resistance may be of great utility in developing commercial clones resistant to this severe disease affecting pepino. This revised version was published online in July 2006 with corrections to the Cover Date.     

Synthesis and preliminary characterization of a new species (amphidiploid) in Cucumis

A successful interspecific hybridization between cucumber (Cucumis sativus L., 2n = 14) and a wild it Cucumis species, C. hystrix Chakr. (2n = 24) was made via embryo rescue. Hybrid plants (2n = 19; 7 from cucumber and 12 from C. hystrix) were sterile, but morphologically uniform. Self-pollination and backcrossing of F₁ hybrid plants to either parent confirmed presence of both male- and female-sterility that were likely caused by lack of homology and improper pairing during meiosis. While the multiple-branching habit, densely brown hairs (on corolla and pistil), orange-yellow corolla, and ovate fruit of F₁ hybrid plants were similar to that of the C. hystrix parent, the appearance of the first pistillate flower was more similar to that of C. sativus parent. The diameter and internode length of the stem, and the shape and size of leaves and flowers were intermediate when compared to the parents. The chromosome number in the hybrid was doubled through somaclonal variation during embryo culture and regeneration process to restore the fertility. Pollen grains were released and fruits with viable seeds matured on fertile, synthetic amphidiploid plants. The results from flow cytometry indicated that, on average, 7.3% of the morphologically unique regenerants had the 4C DNA content of 2.35 pg relative to the 2C DNA content of the original F₁ hybrid at 1.17 pg and, therefore, were likely chromosome-doubled F₁ hybrids (2n = 38). Nutrition alanalysis indicated that the synthetic species had higher protein (0.78%)and mineral (0.35%) content compared to the normal pickling cucumber(0.62% and 0.27%, respectively), and could be considered a new Cucumis crop having a special place in the future agriculture. Preliminarily evaluation indicates that C. hystrix possesses a high level of root-knot nematode resistance, and that this resistance is partially expressed in the interspecific F₁ and chromosome-doubled F₁. This and the fact that the fruit morphology of the fertile amphidiploid differs during the growing season (e.g., short and long fruit) suggest that it could be useful in broadening the germplasm base of cucumber. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chemical Composition and Ruminal Dry Matter and Crude Protein Degradability of Spineless Cactus

The objective of this study was to determine ruminal degradability of the dry matter (DM) and crude protein (CP) of 10 varieties of spineless cactus (Opuntia spp.) grown in north‐eastern Brazil. Two ruminally fistulated steers were used in a randomized complete block design. Ash, CP, acid detergent fiber, and acid detergent lignin levels ranged from 10.4 to 13.3 %, 6.2–7.7 %, 19.8–24.8 % and 3.4–5.4 %, respectively. Relative to the other cactus varieties, Redonda had the highest (P < 0.05) in situ soluble DM fraction and effective DM degradability. No differences in effective DM degradability were observed between the other cactus varieties. In situ soluble CP fraction ranged from 1.7 % of CP for the 69 IPA/UFRPE variety to 11.1 % of CP for the Gigante variety. Slowly degradable CP fraction and its rate of degradation were similar among the cactus varieties (average 90 % of CP and 9 % h^?1, respectively). The cactus variety 69 IP/UFRPE had a lower (P < 0.05) effective ruminal CP degradability (ECPD) than the other varieties, which had a similar ECPD (average 66 % of CP). It was concluded that differences in ruminal degradability exist between cactus varieties, with more variations observed for ruminal DM than for CP degradability.     

Identification of somatic hybrids of dihaploid Solanum tuberosum lines and S. brevidens by species specific RAPD patterns and assessment of disease resistance of the hybrids

Summary Symmetric somatic hybrids were produced by electrofusion of protoplasts of two dihaploid tuber-bearing potato (Solanum tuberosum L.) lines and Solanum brevidens Phil., a diploid non-tuber-bearing wild potato species. A total of 985 plants was obtained. Verification of nuclear hybridity of putative hybrids was based on additive RAPD patterns, general morphological characteristics and chromosome counts. 53 (90%) calli regenerated into plants which were identified as somatic hybrids. Most of the hybrids were aneuploids at the tetraploid (4×) or hexaploid (6×) level. The 20 hybrids tested expressed a high level of resistance to potato virus Y (PVY ^N ) characteristic of the S. brevidens parent. Resistance to late blight (Phytophthora infestans (Mont.) de Bary) varied between hybrids, but was on average better than that of the fusion parents. Resistance of hybrids to bacterial stem rot (Erwinia carotovora subsp. atroseptica (van Hall) Dye) was not superior to that of commercial potato cultivars.     

Enhanced resistance to a lepidopteran pest in transgenic sugar beet plants expressing synthetic <Emphasis Type="Italic">cry1Ab</Emphasis> gene

Two diploid sugar beet genotypes of agronomical importance were transformed using Agrobactrium tumefaciens harboring pBI35Scry containing a synthetic cry1Ab gene. Leaf blade with attached shoot bases, a highly regenerative tissue, were used as explant substratum for transformation. PCR screening with cry1Ab-specific primers showed the presence of transgene in more than 50% of the regenerated kanamycin-resistant plants after treatment with the antibiotic. A transformation rate of 8.8–12.2% (depending on genotype) was achieved as revealed by genomic DNA dot blotting. The intact integration of transgene cassette into the genome was furthermore confirmed by Southern blot analysis. The expression of the cry1Ab gene encoding a truncated endotoxin (67 kDa) at about 0.1% of total soluble protein was achieved in the leaves of transgenic plants as shown by Western blot analysis. Bioassays under in vitro conditions with Spodoptera littoralis, one of the most important pests in sugar beet fields, demonstrated enhanced resistance against this pest. The inheritance of the inserted transgene was confirmed in F₁ plants obtained through crossing of T₀ plants with a cytoplasmic male sterile line. Transgenic plants are currently grown in a greenhouse and will be subjected to further bioassay analyses against other lepidopteran pests of sugar beet.     

Rubus coreanus as donor of resistance to cane diseases and mildew in red raspberry breeding

Summary Strong resistance to the cane diseases Elsinoë veneta, Didymella applanata and Botrytis cinerea, and to Sphaerotheca macularis, occurred in F₁ and BC₁ derivatives of an accession of Rubus coreanus. Resistance to cane spot (E. veneta) was polygenic.In eight out of ten BC₁ progenies, average grades for cane spot infections were significantly higher in white-flowered (an ₁) than pink-flowered (An ₁) plants and in hairy-caned (H) than in glabrous (h) seedlings. It is postulated that in R. coreanus factors controlling resistance are linked with An ₁ and. probably, with h.Average grades for spur blight (D. applanata) were significantly higher in white-flowered plants in nine out of ten BC₁ progenies. Spininess (S) was associated with greater susceptibility in six out of eight BC₁ families, although this difference was not statistically significant.Plants with the phenotype hAn ₁ on average provided the best source of resistance to both cane spot and spur blight.     

Breeding barriers between the cultivated strawberry, Fragaria × ananassa, and related wild germplasm

Five-hundred interspecific and intergeneric crosses were performed among accessions of the wild strawberries Fragaria vesca(2x), Duchesnea indica (8x), Potentilla tucumanensis (2x) and 9 genotypes of the cultivated strawberry, Fragaria×ananassa (8x), following an incomplete diallele mating design. Crosses between D. indica and F.×ananassa produced many putative hybrids when D. indica was used as female but a few achenes and plants when used as male; therefore, pollen-pistil compatibility relations were analyzed by fluorescence microscopy in this direction of the cross. Of the genotypic combinations, 78.6% were incompatible at the stigma level and 17.2% at the first third of the style. Only 3.6% were pollen-pistil compatible and produced fruits with achenes (seven did not germinate or originated short-lived plants and nine produced normal plants). F.vesca×F.×ananassa crosses produced 35 hybrid achenes but only 14% germinated, yielding short-lived plants; histological analyses revealed that inviable seeds had less developed (or collapsed) endosperms and smaller embryos than control plump F. vesca seeds. P.tucumanensis was only used as male, with negative results. These species and genera are partially isolated by a complex system of pre- and post-zygotic barriers. Knowledge of their nature would allow the breeder to devise strategies to put the genetic variability available in the group into a useful form. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of interspecific hybrids between Alstroemeria pelegrina L. var. rosea and A. magenta Bayer by ovule culture

Interspecific hybrids were efficiently produced in the cross-incompatible combination between Alstroemeria pelegrina L. var. rosea and A. magenta Bayer by culturing immature ovules with placenta 7–14 days after pollination on 2 g/l Gelrite-solidified MS medium containing 3% (w/v)sucrose. The plants showed intermediate characteristics between the parents and their hybridity was confirmed by karyotype and DNA analyses. The mean number of chromosome association per PMC at metaphase I was 2.60I+6.70II, pollen stainability was20.8%, and they produced viable seeds after self-pollination. Furthermore, mature plants were obtained when the hybrids were backcrossed as male parents with both the parents. The backcross-progeny from A. pelegrina var. rosea × hybrids exhibited 3.8 to 79.7% pollen stainability and that from A. magenta × hybrids 78.8 to 98.3%. Almost all of these plants produced viable seeds after self-pollination, which implies that they can beutilized for breeding of novel cultivars of Alstroemeria. This revised version was published online in July 2006 with corrections to the Cover Date.     

Resynthesizing <Emphasis Type="Italic">Brassica napus</Emphasis> from interspecific hybridization between <Emphasis Type="Italic">Brassica rapa</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis> through ovary culture

Using three varieties of Brassica rapa, cv. Hauarad (accession 708), cv. Maoshan-3 (714) and cv. Youbai (715), as the maternal plants and one variety of B. oleracea cv. Jingfeng-1 (6012) as the paternal plant, crosses were made to produce interspecific hybrids through ovary culture techniques. A better response of seed formation was observed when ovaries were cultured in vitro at 9–12 days after pollination on the basal MS and B5 media supplemented with 6-benzylaminopurine (BA) and naphthylacetic acid (NAA). The best response was observed for cross 714×6012 with the rate of seeds per ovary reaching 43.0%. Seeds for cross 715×6012 showed the best germination response (66.7%) on the regeneration medium (MS+1.0 mg l^–1 BA+0.05 mg l^–1 NAA). In all three cross combinations, good response in terms of root number and length of plants was observed on the root induction medium (MS+1.0 mg l^–1 BA+0.1 mg l^–1 NAA). A better response was observed for the regenerated plants cultured for 14 days than for 7 days. The ovary-derived plants with well-developed root system were hardened for 8 days and their survival rate reached over 80%. Cytological studies showed that the chromosome number of all plants tested was 19 (the sum of both parents), indicating that these regenerated plants were all true hybrids of B. rapa (n = 10) × B. oleracea (n = 9). The regenerated plants were doubled with colchicine treatment, and the best response in the crosses 708×6012, 714×6012 and 715×6012 was observed when treated with 170 mg l^–1 colchicine for up to 30 h and their doubling frequency reached 52, 56 and 62%, respectively.     

Structural hybridity in the series Etuberosa of the genus Solanum and its bearing on crossability

Summary Three non-tuberous Solanum species of series Etuberosa-S. brevidens (brd), S. etuberosum (etb) and S. fernandezianum (frn)-upon selfing displayed compatible berry set but seed set varied and on an average was below normal. Also in crosses between species berry set was unhampered (apart from the crosses with short-styled frn as pollen plant), but the interspecific hybrid plants showed various degrees of sterility and a low average seed set upon selfing. Cytological investigations revealed that in pure species-plants semi-sterility regularly occurred and was invariably associated with multivalent associations (up to decavalents). This may account for the decreased seed set found after selfing and intercrossing. Meiosis in F₁-hybrids was even more irregular, 75% of the pmc's showing multivalents involving from 4 to as many as 22 chromosomes in various complicated configurations in which even numbers of chromosomes were predominant. However, anaphase I separation was numerically balanced in more than 75% of the cells. Pollen stainability was very low in the hybrids.It is concluded that heterozygous translocations occur within each species and that the genomes of the species are differentiated by means of gross structural changes. In view of this and of the high chromosome pairing affinity the genome symbols E ₁, E ₂ and E ₃ are proposed for etb, brd and frn, respectively.Crossability of the Etuberosa species with tuberous S. pinnatisectum (pnt) was slightly more successful (average 2.7 seeds/berry) than that of Etuberosa species hybrids with S. pinnatisectum (average 1.7 seed/berry). In the hybrids studied-etb × pnt, frn × pnt and (brd × etb) × pnt-a high frequency of univalents was the rule (range 6–16 per cell). In addition rod bivalents (4–8 per cell) and a low frequency of trivalents occurred (0–3 in the biparental hybrids and 1–4 in the trispecific hybrid).Utilization of the valuable characters of Etuberosa for potato breeding is dependent on the degree of gene exchange between Etuberosa chromosomes and chromosomes of tuberous species. In the diploid hybrids such exchange, though limited, may take place through bivalents and trivalents. As shown in this article such diploid hybrids are non-crossable because of complete male and female sterility. Only after doubling the number of chromosomes the hybrids are fertile and can be used for further crosses.     

Particle-Bombardment-Mediated Co-Transformation of Maize with a Lysine Rich Protein Gene (sb401) from potato

Summary Little work has been reported on genetic transformation with maize inbred lines, especially elite inbred lines used in breeding. In this work, 7 self-pollinated inbred lines and 4 hybrid lines have been screened. The results revealed that calli derived from immature embryos from two inbred lines X333 and X301 were compact, hyperhydric and unsuitable for transformation, but the calli induced from other inbred lines and all the hybrid lines were friable and yellow and could be used for genetic transformation. The sb401 gene isolated from potato (Solanum berthaultii) encodes a protein with a high lysine content. Maize calli from 5 self-pollinated inbred lines and 4 hybrid lines were transformed using particle-bombardment with different plasmids to simultaneously introduce the sb401 lysine rich gene and the selectable gene hpt respectively. Two hundred and sixty-eight regenerated plants were obtained from these genotypes. Co-insertion was confirmed in 29 regenerated plants by PCR and Southern blot analysis. Transgene segregation of the R1 plants was observed and one marker-free transgenic maize line was recovered. Analysis of the crude protein content in mature seeds of R1 transgenic plants also showed an increase from 36.8% to 48.2%. This study thus provides a workable system for generating transgenic maize free from selectable marker genes and generates valuable resources for obtaining marker free transgenic maize with a high-lysine protein content.     

Application of in vitro organ culture in wide-cross breeding of rapeseed

Oil radish (Raphanus sativus var. raphanistroides Makino) is resistant to drought and low temperature. In order to breed more resistant cultivars of rapeseed, the wide cross between rapeseed (Brassica napus L.) and oil radish was made. Rapeseed was not compatible with oil radish, and the frequency of hybrid plants (F₁) was very low. Moreover, the hybrid plants were sterile. In order to recover the intergeneric hybrids (F₁), the in vitro organ culture technique was applied in our experiments. The frequency of hybrid plants (F₁) was increased up to 25.55% by means of in vitro culture of pollinated ovaries. Some fertile amphidiploid hybrid plants were obtained by means of colchicine treatment of small buds obtained from cultured flower receptacle segments of hybrid plants (F₁). It is suggested that the technique of in vitro culture of pollinated ovaries and flower receptacle segments is useful in the wide-cross breeding of rapeseed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ploidy analysis in water yam, Dioscorea alata L. germplasm

Poor reproductive development in yams (Dioscorea spp.) has often been attributed to the polyploid nature of the crop. In this study, flow cytometry was used to determine the ploidy level of 53 accessions of Dioscorea alata, mostly from West African countries, Chad and Puerto Rico. Nuclei were isolated from young leaf material and stained with DAPI(4,6-diamidino-2-phenylindole). The nuclear genome size (2C) was measured as an indicator of the ploidy level. Dioscorea rotundata genotypes with known ploidy levels were used as standards. The results showed that the majority of plants were hexaploid (84.9%) with a smaller percentage of tetraploids (15.1%). A higher number of male plants were hexaploid than tetraploids. This is at variance with earlier findings, which reported that hexaploid male plants are rare. Higher ploidy levels were not directly related to sparse or erratic flowering as previously reported as profuse flowering occurred in some male hexaploid accessions. These findings have important implications for yam breeding in relation to yam genetic resources. This revised version was published online in August 2006 with corrections to the Cover Date.