New types of major anthocyanins detected in Japanese garden iris and its wild forms

The anthocyanins of 130 cultivars, 13 lines and 3 wild forms of Iris ensata were analyzed by HPLC, and these plants were classified into 16 types of major anthocyanins. Among these types, 8 types such as petunidin 3RGac5G – delphinidin 3RGac5G, delphinidin 3RGac5G – petunidin 3RGac5G, cyanidin 3RGac5G – peonidin 3RGac5G, delphinidin 3RG – delphinidin 3RGac, petunidin 3RG5G – malvidin 3RG5G, malvidin 3RG5G – peonidin 3RG5G, peonidin 3RG5G – cyanidin 3RG5G and peonidin 3RG – cyanidin 3RG were obtained as new types. In these new types, peonidin 3RG – cyanidin 3RG and peonidin 3RG5G – cyanidin 3RG5G types were noteworthy because cyanidin 3RG and cyanidin 3RG5G are useful for the breeding of red flowers in I. ensata. This revised version was published online in July 2006 with corrections to the Cover Date.     

Overcoming the barrier to interspecific hybridization of Fagopyrum esculentum with Fagopyrum tataricum

Summary Tartary buckwheat (Fagopyrum tataricum) was successfully hybridized with common buckwheat (F. esculentum), both diploid (2n=16), using the latter as male parent during bud pollination. The barrier normally encountered in such hybridization was overcome by enhancing the cross-compatibility of the two species, which was accomplished by synthesizing a unique genotype of common buckwheat. This novel plant was produced by selecting common buckwheat plants that exhibited, at isozyme loci PGM, SKDH and ADH, alleles with similar mobility to those found in tartary and then transferring these alleles to a single plant through six generations of breeding. Ovule culture was used to rescue the 7–10 day old embryos. On the rescue-culture medium 41% of the hybrid embryos formed calli larger than 200 m in diameter. Most ceased to grow before reaching 1500 m, but four out of 263 cultured ovules continued to grow as callus. One of these differentiated and formed callus with buds and shoots from which cloned plants were produced. The remaining embryos either did not grow at all or formed very small calli. When tartary was crossed with the original genotype of common buckwheat, only 22% of the hybrid embryos formed small calli and none differentiated. Hybridity of the calli and the plantlets was determined by RAPD and isozyme analysis, respectively. Flowers produced by the hybrid plants were of the same type (homomorphic) and size as those of tartary, but with white sepals like common buckwheat.Abbreviations CTAB cetyltrimethylammonium bromide - GA₃ gibberellic acid - IAA indoleacetic acid - IBA indolebutyric acid - PVP polyvinylpyrrolidone     

High-performance liquid chromatographic analysis of anthocyanins in induced amphidiploids of Iris laevigata Fisch. × I. ensata Thunb.

Summary Anthocyanins of colchicine-induced amphidiploids of I. laevigata × I. ensata were analyzed by the HPLC procedure and compared with those of parental species and F₁ hybrids of I. laevigata × I. ensata. The amphidiploids were characterized by seventeen anthocyanins, and exhibited all anthocyanins of the parental species and the F₁ hybrids. Malvidin 3RGac5G and petunidin 3RGac5G in anthocyanins detected were major pigments in the amphidiploids, which exhibited strongly the malvidin 3RGac5G-petunidin 3RGac5G type of I. ensata rather than the petunidin 3RGac5G-malvidin 3RGac5G type of I. laevigata. Moreover, a higher degree of anthocyanin content was observed in the amphidiploids and the F₁ hybrids. Namely, the anthocyanin content of the amphidiploids and the F₁ hybrids were 2.81 and 2.45 times as much as that of the mid-parent. This implies that there is a complementary effect on anthocyanin synthesis of parental genes in them. Finally, the utility of the amphidiploids was discussed.Contribution from the Laboratory of Plant Breeding, Faculty of Agriculture, Miyazaki Univ., No. 64.     

Inter-varietal variations of rutin content in common buckwheat flour (Fagopyrum esculentum Moench.)

Summary To improve the quality of buckwheat flour, we investigated the effect of the cropping season on the rutin content in various cultivars of common buckwheat (Fagopyrum esculentum Moench.). Thirty cultivars of buckwheat were grown under both long day conditions (the summer cropping) and short day conditions (the late summer cropping). The inter-varietal variations and the effect of the cropping season on the rutin content were examined. The rutin content was determined by high performance liquid chromatography (HPLC). Results show that the average rutin content in the late summer cropping was about half that of the summer cropping. There were wide inter-varietal variations of rutin content in the summer cropping. The differences in rutin content in the late summer cropping were less pronounced. Rutin content was closely correlated with the flowering period in summer (r=0.735^***). The later flowering cultivars produced a higher content of rutin than the earlier flowering cultivars in the summer cropping. The differences in the cumulative solar radiation received in the two seasons seems to be a reasonable explanation for this increase in the rutin content of the buckwheat flour. The rutin content of early flowering cultivars, which are suitable for long day conditions, is positively correlated with yield in the summer cropping. However, the rutin content in the intermediate and late summer ecotype cultivars, which are suitable for short day conditions, shows no correlation with yield in the late-summer cropping. In conclusion, there are wide inter-varietal variations of rutin content in buckwheat flour and the rutin content increases under long day conditions.     

In vitro propagation of Japanese garden iris,Iris ensata Thunb.

Summary Explants of young scapes of Iris ensata were cultured on MS medium with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar, and this species was characterized by high variety specificity for callus, shoot and root induction. Among 23 varieties and one wild form tested, Okichidori, Miyukisudare and Meiji-l exhibited a considerable rate of shoot induction, although these induced poorly rooted shoots. In addition, two types of callus induction such as green and white calli were observed, and the induction of green-type calli was significantly correlated with that of shoots. Surprisingly, the only modification, half-strength MS inorganic salts, for the above medium proved to be very effective for shoot induction in the scape culture. For shoots obtained from the scape culture, effects of sucrose concentrations and activated charcoal on root induction were examined by using 1/2 MS with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar as the basic medium. The addition of 1% activated charcoal to the media had a marked effect for root induction independent of sucrose concentrations and varieties tested. The in vitro propagation technique of I. ensata is discussed.     

Random amplified polymorphic DNA (RAPD) variation in Fagopyrum tataricum Gaertn. accessions from China and the Himalayan region

The diversity among 52 landraces and cultivars of tartary buckwheat (Fagopyrum tataricum Gaertn.) and one accession of its wild ancestor, F. tataricum ssp. potanini Batalin, from diverse geographic origins was examined using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers. Eighteen primers produced a total of 240 fragments, of which 153 (63.75%) were monomorphic and 87 (36.25%) polymorphic bands. UPGMA-based pairwise Jaccard’s coefficient of similarity was used to deduce the relationships among 53 genetically diverse accessions. The similarity between cultivated tartary buckwheat accessions ranged from 0.61 to 1.00. Four distinct clusters were formed which corresponded well with the geographic distribution of the tartary buckwheat. Nepalese accessions showed maximum diversity followed by Chinese accessions. Tartary buckwheat accessions from the Himalayan region of northwestern India revealed a narrow gene pool. The wild buckwheat accession did not group with any of the three cultivated tartary buckwheat groups, and formed its own single-entry group. Genetic similarity (0.59) of Chinese buckwheat accessions with the wild ancestor reaffirmed that cultivated tartary buckwheat originated in the Yunnan province of northwestern China. Consistent with some earlier reports, our study demonstrated the usefulness of the RAPD technique for the characterization of plant genetic resources and assessment of diversity between species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Use of ovule culture to cross between Delphinium species of different ploidy

The applicability of ovule culture was examined in reciprocal crosses between the diploid species D. grandiflorum L. and the tetraploid cultivar D. hybridum ‘Galahad’. An interspecific hybrid plant of D. hybridum ‘Galahad’ × D. grandiflorum was obtained,and hybridity was confirmed by chromosome counting. The hybrid was different from the parents in flower color and size but intermediate between the parents in leaf shape. The hybrid showed low pollen fertility and failed to produce viable seeds by either self- or backcross-pollination using fertile pollen grains from its parents. The applicability and efficiency of the ovule culture technique for genetic improvement of the genus Delphinium through interspecific hybridization between different ploidies is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cross compatibility between chickpea and its wild relative,Cicer echinospermum Davis

Summary Cicer echinospermum, a wild relative of chickpea (Cicer arietinum L.), has traits that can be used to improve the cultivated species. It is possible to obtain successful crosses between the two species, even though their cross progenies have reduced fertility. The reasons for this low fertility could be due to the two species differing in small chromosome segments or at genic level. Another limitation to the use ofC. echinospermum at ICRISAT Asia Center is that the species is not adapted to the short photoperiod which prevails during the chickpea cropping season at Patancheru, Andhra Pradesh, India. Future work will include screening the segregating progenies for monitoring traits from both the species through isozyme analysis and to incorporate these into good agronomic backgrounds following backcrosses.Submitted as JA 1669 by ICRISAT     

Interspecific Hybridization in the Genus Lens Applying in Vitro Embryo Rescue

Summary Four different hybridization experiments were carried out to obtain interspecific hybrids with Spanish cultivars of lentil (Lens culinaris M.). In hybridization experiments I and II, undertaken with only pollination and pollination with the addition of gibberellic acid after fertilization, respectively, no lentil hybrids were recovered. A single interspecific hybrid with L. odemensis was obtained in experiment III using the embryo rescue protocol of Cohen et al. (1984), in this case, a crossing efficiency of 0.11% and a rescue efficiency of 2.5% were obtained. Hybridization experiment IV used a specific embryo rescue protocol developed in this study. In this experiment, ovule-embryos of 18 DAP were cultured on MS salts with 1% sucrose and 1 μ M IAA + 0.8 μ M KN; after two weeks, embryos were released from the ovular integuments and cultured on the same medium for another two weeks in upright position. Afterwards, the embryos were transferred to test tubes containing the same medium and one month later plantlets were obtained. Using the above protocol, out of a total of 1707 pollinations, 6 interspecific hybrids with L. odemensis, 2 with L. nigricans and one with L. ervoides were recovered, yielding on average a crossing efficiency of 0.53% and an average rescue efficiency of 8.26%. Taking into consideration only the interspecific crossing blocks in which hybrids were recovered, the crossing efficiency with L. odemensis was 9%, while with both L. nigircans and L. ervoides the crossing efficiencies were 3%. Rescue efficiencies based on hybrids recovered per number of ovules cultured ranged between 50–100%.     

Embryo-rescue in the genus Tulipa L.; successful direct transfer of T. kaufmanniana Regel germplasm into T. gesneriana L.

Summary Embryo-rescue was studied as a means to overcome post-fertilization barriers in interspecific crosses in the genus Tulipa. With compatible T. gesneriana L. cultivar crosses, ovule culture was found to be superior to isolated embryo culture. Complete plantlet formation was possible from an embryo size of about 0.5 mm onwards.In the interspecific cross T. gesneriana x T. kaufmanniana, which is hampered by embryo breakdown, successful rescue of abortive embryos was demonstrated. Optimal embryo-rescue was achieved in cultures started seven to nine weeks after pollination. With cultures initiated at a later time, the rate of success decreased. A low number of germinative seeds were obtained after normal ripening of the seed pods, but by using ovule culture the efficiency of seedling formation could be increased dramatically.The ovule culture procedure will allow novel crosses and will offer new possibilities for the introduction of desirable genes into tulip cultivars.     

Seed protein variation for identification of common buckwheat (Fagopyrum esculentum Moench) cultivars

Summary Total seed proteins of 24 common buckwheat cultivars and cultivated populations within a molecular weight range of 30 to 54 kDa were analysed by SDS-PAGE. Single seed analysis of six cultivars identified a total of 18 alternative protein bands with different mobilities. Differences of individual protein band frequencies extracted from single seeds among six buckwheat cultivars varied distinctively, indicating high intravarietal polymorphism. The relation between frequencies of protein bands revealed by single seed analysis and their appearance on the bulk seed analyses was demonstrated. Regarding to band mobility rate and relative band intensity among 24 bulk samples analysed, 14 had distinctive electrophoregrams while the other 10 were ranged into four distinct groups. Analysis of endosperm and cotyledon proteins showed that proteins stored in these main seed parts are tissue specific. The observed electrophoretic polymorphism related to proteins stored in the cotyledons while there was no apparent variability with endosperm proteins.     

Variation in pollen performance among plants of Fagopyrum esculentum

Summary In genetically-heterogeneous outcrossing species, there is the opportunity for selection based on the male gamete. Buckwheat (Fagopyrum esculentum Moench) is self-incompatible with one ovule per flower, so pollen competition at each ovule can be studied. The occurrence of selection among pollen parents was determined, as well as the relative importance of prezygotic and post-zygotic selection. Mixed pollinations from two donors produced nonrandom paternity, with one of the donors being favored about 2:1 on several females. Individual plants showed significant variation in the speed of pollen-tube growth. Therefore, prezygotic selection is likely to have occurred based on the speed of pollen tube growth. In single-donor pollinations, donors had equal success as expected in the absence of post-zygotic selection among donors. However, a significant male x female interaction was found, consistent with postzygotic selection against particular parental combinations. To test whether male fitness is reduced by increased allocation to seed filling during pollen production, large- and small-seeded lines were compared, both as pollen donor and as pollen recipient. The large-seeded line was better in both roles, thus there was no evidence that greater allocation to seeds reduced the quality of the pollen.     

Hybridization between diploid (Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton through ovule culture

Summary With in vitro culture of ovules, interspecific hybrids have been obtained in an otherwise incompatible cross between a diploid (Gossypium arboreum) and a tetraploid (G. hirsutum) cultivated cotton. The early abortion of the embryo was prevented by repeated treatment of the flowers, immediately after pollination with a solution of gibberellic acid and naphthalene acetic acid. The ovules excised three days after pollination and cultured in a liquid medium underwent profuse proliferation, whereas on an agar-solidified medium supplemented with casein hydrolysate, indoleacetic acid and kinetin they germinated to form hybrid plants.     

Karyotypes and chromosome association in aneuploid varieties of Japanese garden iris,Iris ensata Thunb

Summary Karyotypes, chromosome association and pollen fertility of aneuploid varieties (2n=25), Ochibagoromo, Matsusakatsukasa and Isehomare in Iris ensata were analysed and compared with those of eu-diploid varieties (2n=24), Shishinden, Kachô and Asahimaru. The somatic chromosome complement of the aneuploid varieties consisted of 11 pairs and 3 singles of chromosomes and that of the eu-diploid varieties 12 pairs of chromosomes. The singles of chromosomes in the aneuploid varieties had similarity with one another and with a pair of chromosomes in the eu-diploid varieties. The high frequency of normal association was present in the eu-diploid varieties, and this indicated that 12 pairs of chromosomes had full homology between each other. In contrast, the mean chromosome association per cell in an aneuploid variety Ochibagoromo was 4.615_I+10.067_II+0.077_III+0.005_IV, indicating that the chromosome complement of this variety consisted of 11 pairs and 3 singles and that these singles had partial homology among them. The eu-diploid varieties exhibited high pollen fertility due to their regularity of chromosome association; the aneuploid varieties considerably lower fertility, i.e. 28.2% for Ochibagoromo, 31.8% for Isehomare and 43.8% for Matsusakatsukasa. The primary cause for the low fertility of these varieties seemes to be the partial homology among 3 single chromosomes. Finally, the origin and the development of the aneuploid varieties were discussed.     

Interspecific hybrids between three eggplant (Solanum melongena L.) cultivars and two wild species (Solanum torvum Sw. and Solanum sisymbriifolium Lam.)

Three Greek eggplant cultivars, ‘Langada’, ‘Tsakoniki’ and ‘Emi’ (2n= 24), were crossed with two wild species (Solanum torvum Sw., 2n= 24 and Solanum sisymbriifolium Lam., 2n= 24). Ovules isolated 15-27 days after pollination were cultured in a modified MS medium at 24°C and a 16h photoperiod. Fifty days later, the ovules were dissected and the interspecific embryos were cultured in the same medium. Interspecific hybrids were achieved only from crosses between the eggplant cultivars and S. torvum. The hybridity of the putative interspecific F¹ hybrid (Solanum melongena×S. torvum) was confirmed by using morphological and biochemical (isozyme isocitrate dehydrogenase A, phosphoglucomutase A, phosphoglucose isomerase B, 6-phosphogluconate dehydrogenase A, 6-phosphogluconate dehydrogenase B) markers. The F¹ plants (‘Langada’×S. torvum) were selfpollinated and backcrossed to both parents. Fruits, however, were produced only when the F¹ hybrid was backcrossed as female with the eggplant cultivar ‘Langada’.     

Phylogenetic analysis of AGAMOUS sequences reveals the origin of the diploid and tetraploid forms of self-pollinating wild buckwheat,Fagopyrum homotropicum Ohnishi

Fagopyrum homotropicum Ohnishi is a self-pollinating wild buckwheat species indigenous to eastern Tibet and the Yunnan and Sichuan Provinces of China. It is useful breeding material for shifting cultivated buckwheat (F. esculentum ssp. esculentum Moench) from out-crossing to self-pollinating. Despite its importance as a genetic resource in buckwheat breeding, the genetic variation of F. homotropicum is poorly understood. In this study, we investigated the genetic variation and phylogenetic relationships of the diploid and tetraploid forms of F. homotropicum based on the nucleotide sequences of a nuclear gene, AGAMOUS (AG). Neighbor-joining analysis revealed that representative individuals clustered into three large groups (Group I, II and III). Each group contained diploid and tetraploid forms of F. homotropicum. We identified tetraploid plants that had two diverged AG sequences; one belonging to Group I and the other belonging to Group II, or one belonging to Group II and the other belonging to Group III. These results suggest that the tetraploid form originated from at least two hybridization events between deeply differentiated diploids. The results also imply that the genetic diversity contributed by tetraploidization of differentiated diploids may have allowed the distribution range of F. homotropicum to expand to the northern areas of China.     

Effects of cultivar and environment on phenolic acids in buckwheat

Summary Phenolic acid contents were determined in seeds from five buckwheat cultivars grown at three locations in western Canada for four years. Buckwheat contained 12–16 g/kg of total phenolic acids, about 3 g/kg of esterified phenolic acids, and 8–13 g/kg of etherified phenolic acids. The latter represented 70 to 79% of the total phenolics. Variation in phenolic acids was mainly due to cultivar, seasonal effects and their interaction, while growing location had no significant effect. Phenolic acid contents of buckwheat were independent of seed color and protein content. Esterified phenolic acids exhibited a negative significant association with etherified phenolic acids in buckwheat.     

Stability of flower colors due to anthocyanin-flavone copigmentation in Japanese garden iris, Iris ensata Thunb.

The fading of flower color in bluish purple and reddish purple cultivars of Iris ensata and the in vitro stability of malvidin 3RGac5Gand petunidin 3RGac5G due to copigmentation with isovetixin under different pH conditions were examined. The bluish purple cultivars exhibited higher flower color stability than the reddish purple cultivars 2 days after anthesis. In the absence of isovitexin, malvidin 3RGac5G and petunidin 3RGac5G were not able to maintain color stability except at low pH. However, the color stability of malvidin 3RGac5G and petunidin 3RGac5G was increased by copigmentation with isovitexin under all pH conditions tested. Most remarkable was the stabilization of both anthocyanins due to the copigmentation at pH 4.2–6.2. Therefore, it can be concluded that the stability of flower color in the bluish purple cultivars of malvidin 3RGac5Gand petunidin 3RGac5G type of I. ensata is caused at least in part by the copigmentation between these anthocyanins and isovitexin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interspecific hybridization between rice bean (Vigna umbellata) and its wild relative (V. minima): Fertility-sterility relationships

Summary The pre-and post-fertilization barriers in the interspecific crosses between Vigna umbellata and V. minima were investigated. In the reciprocal crosses (V. minima as the parent) the entry of pollen tubes into the ovary was delayed by about 4 h, and no seed set was observed. However, no pre-fertilization barriers were encountered in crosses involving V. minima as the parent and V. umbellata as the parent (normal cross). The delay/absence of divisions in the endosperm and the failure of embryo to divide were the post-fertilization barriers responsible for somatoplastic sterility in normal crosses which yielded a few hybrid seeds. The hybrid seeds showed poor germinability. The F₁ hybrids were intermediate between the parents in most morphological characters, and are completely sterile for pollen. Backcrossing of F₁ hybrid with both the parents did not restore fertility in the progenies. V. minima is considered as the tertiary gene pool of the rice bean.     

The origin of lentil and its wild genepool

Summary Following hybridization experiments and cytogenetic analysis of interspecific hybrids three chromosome interchanges were found between the cultivated lentil L. culinaris and L. nigricans, and only one between the cultivated species and L. orientalis. This indicates that the latter species is more likely to be wild progenitor of lentil. The partial fertility of the interspecific hybrids indicate further that both L. nigricans and L. orientalis should be included in the wild genepool of lentil, and their variation can be exploited by relatively simple hybridization techniques. The wild lentils L. orientalis and L. nigricans are morphologically very similar but reproductively strongly isolated from one another by the albino seedling of their hybrids. It has been suggested that the populations of L. orientalis that gave rise to the cultivated lentil still possess a similar chromosome arrangement as in L. culinaris and are also capable of forming normal hybrids with L. nigricans. According to these considerations it is unlikely that lentil originated from populations at the south western corner of the distribution area of L. orientalis.