A new system for simple and efficient shoot regeneration of soybean (Glycine max (L.) merr.) is reported using the hypocotyl of mature seeds. Two transverses of 1-mm sections of the hypocotyl were cut from mature seeds: sections containing a cotyledonary node and sections 1 to 2 mm from the cotyledonary node. The effects of thidiazuron (TDZ) concentrations, plating methods, and genotypic differences were examined. Shoots were formed from cotyledonary node ends in all conditions examined. Meanwhile, shoots were formed from the ends of hypocotyl sections on B5 media containing TDZ. The optimal TDZ concentration for organogenesis was 2~10 μM. The efficiency of organogenesis varied with the method of plating of explants. The ends of the hypocotyl sections in contact with the media only produced adventitious shoots. Adventitious shoots were formed effectively by placing explants on the media oriented so that the hypocotyl axes were perpendicular to the surface of the media. The efficiency of organogenesis differed with the position of the hypocotyl ends. It was important to use the ends that were 1 mm from the cotyledonary nodes. Genotypic differences were observed in the organogenesis of the hypocotyl ends.
To obtain a desirable hybrid from an interspecific cross between Fagopyrum tataricum and F. esculentum, the early embryo developmental changes were investigated by light microscopy and transmission electron microscopy (TEM). The nature of pre- and post-fertilization barriers in the hybrid embryo was examined at the ultrastructural level. The ovules were excised at 1 to 5 days after pollination (DAP) and fixed for TEM. In comparison with self-pollinated embryo (F. tataricum) or legitimate-pollinated embryo (F. esculentum), the interspecific hybrid embryos showed numerous abnormal ultrastructural pre- and post-fertilization phenomena leading to the abortion of embryo. The main abnormal phenomena were failure of fertilization, no zygotic development with a broken cell wall, dispersed endosperm with absence of ribosomes, and defective embryo sac development such as srunken, small and irregular-shaped embryo sac. The interspecific incompatibility was attributed to the failure of fertilization and the hybrid zygotic embryo showed retardation in cell division, which resulted in cease of development.
In order to determine whether anther-derived varieties and conventional varieties behave in the same manner as theoretically assumed, the stability of the characters in a doubled haploid variety Shirayukihime, developed by anther culture of rice (Oryza sativa L.), was evaluated compared with three varieties, Koshihikari, Nipponbare and Hatsushimo, developed by conventional methods of breeding. Days to heading, culm length, panicle length and panicle number of those varieties were measured or counted generation by generation for ten years. The days from the beginning to the end of heading were not different between the doubled haploid variety and the conventional varieties. The stability of culm length and panicle length was not different among the four varieties based on the non-significant difference in their coefficients of variation (CV). The CV in culm length was found to be highly stable, as no significance was detected for year-to-year and variety-to-variety. In the CV of panicle numbers, significant differences were detected in both years and varieties, between early and late plantings. However, no significant difference was detected between the doubled haploid variety and the conventional varieties in both plantings. Therefore, the CV in the characters of Shirayukihime were not higher than those of the other varieties. These results suggest that the stability of the characters of the doubled haploid variety might not be different from those of conventional varieties. The conventional method of breeding could thus be efficiently supplemented with the anther culture method.
The effect of low temperature pretreatment of buds or inflorescence on microspore culture for the production of haploids of Brassica rapa (syn. B.campestris) was examined. Incubation of the buds or the inflorescence at 4°C for 3 or 10 days before the culture of microspores induced efficient microspore embryogenesis. Pretreatment of flower buds was more effective than that of the inflorescence. Prolonged pretreatment up to 20 days promoted embryo induction. Microspores in the buds were examined for developmental stage before and after the pretreatment. Buds with a petal length to anther length ratio of about 0.5-0.7, which had microspores were at the late unicellular stage, were collected and were used. All microspores were at the late unicellular stage before the pretreatment. The percentage of the microspores at the stage of late unicellular decreased during the pretreatment while the percentage of bicellular stage microspores with two unequal size nuclei increased. At the same time, a small but notable number of bicellular stage microspores with equal size nuclei, which is the first step of microspore embryogenesis, was observed after the pretreatment.
Alstroemeria ligtu L. hybrid (LH, 2n = 2x = 16), A. pelegrina L. var. rosea (PR, 2n = 2x = 16) and their interspecific hybrids (2n = 2x = 16, 3x = 24, 4x = 32) were analyzed cytologically. In the parental species, the mean chromosome association per pollen mother cell (PMC) at metaphase I in meiosis was 0.04 I + 7.98 II in LH and 0.08 I + 7.96 II in PR. Meiosis proceeded normally in these 2 species and pollen fertility was 98.4 % in the former and 94.9 % in the latter. These 2 species produced many normal seeds by self-pollination. The hybrid (2n = 16) showed partial chromosome association with a mean of 11.18 I + 2.41 II. Abnormal chromosome behavior such as the formation of bridges and micronuclei was observed frequently at anaphase I and II of PMCs or at the early uninucleate stage of microspores, which resulted in low pollen fertility (0.6 %). The hybrid did not produce any normal seeds when self-pollinated or backcrossed with either parent. In the amphidiploid (2n = 32), the mean chromosome association per PMC was 0.82 I + 15.59 II at metaphase I and pollen fertility was 86.3 %. Although the amphidiploid did not produce any normal seeds by self-pollination, it produced normal seeds by the reciprocal crosses with LH. The mean chromosome association in the sesquidiploid (2n = 24) of LH × the amphidiploid was 8.24 I + 7.85 II + 0.02 III and that of PR × the amphidiploid was 8.58 I + 7.66 II + 0.03 III. These sesquidiploids frequently showed chromosome bridges and micronuclei at anaphase I and II of PMCs or at the early uninucleate stage of microspores, and pollen fertility was 14.8 % in the former and 13.0 % in the latter. Although the two types of sesquidiploid did not produce any normal seeds by self-pollination, the former sesquidiploid produced normal seeds by the cross with LH. In LH, the Giemsa C-bands were observed in 7 out of 8 bivalents at metaphase I of PMCs and in 7-7 out of 8-8 chromosomes at anaphase I of PMCs, whereas no C-bands were observed in PR throughout these meiotic stages. These C-band patterns were detected at metaphase I of PMCs in the hybrid, the amphidiploid, and the two types of sesquidiploids.
A new quantitative trait locus (QTL), Hd9, controlling rice heading date was detected in a BC4F2 backcross population derived from a cross between a japonica rice variety, Nipponbare, as the recurrent parent, and an indica variety, Kasalath, as the donor parent. Hd9 was precisely mapped on the short arm of chromosome 3 as a single Mendelian factor; progeny testing of BC4F3 lines derived from BC4F2 plants revealed that it co-segregated with the RFLP marker S12021. In addition, a nearly isogenic line (NIL) of the target QTL, NIL(Hd9), in which a small chromosomal segment of Kasalath including Hd9 was substituted into the genetic background of Nipponbare, was selected from the progeny based on marker-assisted selection. Days to heading of NIL(Hd9) increased under long-day and natural field conditions at Tsukuba, but remained almost the same under short-day conditions as that of the isogenic control, Nipponbare. Analysis of F2 populations derived from crosses between NIL(Hd9) and either NIL(Hd1) or NIL(Hd2) revealed that the phenotypic gene effect of Hd9 was additive to that of Hd1 and Hd2. This result suggests that no epistatic interaction was involved. Based on these results, we discussed the biological function of Hd9.
We introduced a truncated delta-endotoxin gene, cry1Ab of Bacillus thuringiensis which has a specific biological activity against lepidopteran insects into chrysanthemum [Dendranthema × grandiflorum (Ramat.) Kitamura]. The chrysanthemum cultivar Shuho-no-chikara was transformed using a disarmed strain of Agrobacterium tumefaciens, LBA4404, carrying a binary vector, pIAbT1 that harboured a cry1Ab gene encoding an insecticidal crystal protein fragment of B. thuringiensis var. kurstaki HD-1. Leaf discs were co-cultured with Agrobacterium and thereafter cultured on the callus induction medium containing G418. A total of 92 shoots were regenerated from 1,760 leaf discs on the regeneration medium (5.2 %). The cry1Ab gene was detected in all the regenerated plantlets by Southern blot analysis. The accumulation of Cry1Ab protein in 20 transformed lines, selected at random, was confirmed by Western blot analysis. The level of accumulation of Cry1Ab protein ranged from 4.5 ng to 40 ng per 50 μg total soluble protein (from 0.009 to 0.08 % of the total protein). Insect bioassay was conducted using tobacco budworm (Helicoverpa armigera) larvae. On the lines showing a high expression of Cry1Ab (more than 32.5 ng per 50 μg of total soluble protein), a significantly higher feeding inhibition and/or growth inhibition of the insects was observed, compared to those on the non-transformed control plants.
To produce intergeneric hybrid plants with one or a few chromosomes via microprotoplast fusion in the Liliaceous ornamentals, we developed a system for isolating microprotoplasts from micronucleated cells in suspension cultures of Hemerocallis hybrida cv. Stella dOro (2n = 2x = 22). Micronucleation was efficiently induced with a micronucleus index up to 19.1 % by the following sequential treatment of suspension cultures: initially with 2 mM hydroxyurea for 24 h and then with 8 μM propyzamide for 60 h with the addition of 20 μM cytochalasin-B to the cultures 20 h after the initiation of the propyzamide treatment. Following the enzyme treatment of the micronucleated cells and ultra-centrifugation with a continuous iso-osmotic gradient of Percoll solution, microprotoplasts were isolated, each of which had a small nucleus surrounded by a thin rim of cytoplasm. Microprotoplasts less than 10 μm in diameter were obtained with yield of 2.9 ×10 4 cells per 1 ml packed cell volume of suspension cells through sequential filtration with nylon sieves with decreasing pore sizes (50, 20 and 10 μm). The DNA content of the microprotoplasts was less than the 2C level as indicated by flow cytometric analysis, and the relative fluorescence intensity in some of their nuclei corresponded to one or a few chromosomes.