Stems of Mains prunifolia Borkh. var. ringo Asami strain Nagano No.l were infected with Agrobacterium rhizogenes MAFF-02-10265, MAFF-02-10266, MAFF-03-01724 and MAFF-03-01725 by using a needle. The frequency of hairy root formation was 2.0% with both MAFF-02-10265 and MAFF-02-10266. Transgenic plants were obtained from hairy roots that were induced by A. rhizogenes MAFF-02-10265. Adventitious shoots were formed from the hairy roots on Murashige and Skoog medium, supplemented with 5.0 mg·liter-1 6-benzyl-aminopurine (BAP), 0.1 mg·liter-1 indole-3-butyric acid (IBA) and 0.1 mg·liter-1 gibberellic acid (GA3). These shoots developed abundant roots in the culture medium. The transgenic nature of the regenerated plants was confirmed by opine and PCR analysis. A transgenic plant, acclimatized in the greenhouse, showed prolific branching, miniaturized leaves, and shortened internodes.
The 84 cross combinations used in the Japanese pear breeding program in 1953-1955 were investigated to identify the parents of 'Housui' (syn. 'Hosui'). Twenty-one putative parent cultivars were investigated for their S genotypes and skin types. The S genotypes of 75 out of 84 cross combinations could not have sired 'Housui' (S3S5) because neither putative parent has an S3 nor S5 allele. Skin types of all cultivars used for 9 cross combinations selected from S genotype analysis were russet types. For this reason, 9 cross combinations are possible sources for the putative parents of 'Housui'. These 9 cross combinations were investigated by utilizing 10 SSR markers developed from Japanese and European pears. The SSR analysis indicated that only two parental crosses, 'Kousui' (syn. 'Kosui') × '1-33' and '1-33' × 'Kousui' were possible; the other 7 parentages had discrepancies at the 2-6 loci. The two assumed parents of 'Housui', 'Kousui' and 'I-33' were investigated by using 51 more SSR markers. A total of 61 SSR marker analyses confirmed the parent-offspring relationships because 'Housui' inherited SSR alleles from 'Kousui' and '1-33' without any discrepancy. Chloroplast DNA (trnL-F region) was investigated to determine whether 'Kousui' or '1-33' was the female parent. The nucleotide sequence at the trnL-F region of 'Housui' was exactly the same as that of 'Kousui', but was different from that of '1-33'. Therefore, we conclude that 'Kousui' is the female parent of 'Housui' and '1-33' is the male parent.
Chill units (CU) and growing degree hours in Celsius (GDH°C) required for normal flowering with flower buds on excised shoots that were collected from the field during early December 2002-late February 2003 and forced under a dark frame at 20°C. CU varied with cultivars, ranging from 732 in 'Okinawa' to 1433 in 'Kikumomo'. GDH°C also varied with cultivars under forced and natural conditions. CU was negatively correlated with forcing GDH°C except for 'Genpeishidare'. Prolonged CU after breaking endodormancy (rest) reduced GDH°C under forced conditions. CU was positively correlated with the mean flowering dates at 20% full bloom (FB) (r=0.87, P ≤ 0.001) and 80% full bloom (r=0.81, P ≤ 0.01). There were significant differences in GDH°C required for 20% and 80% FB under natural conditions among cultivars; their correlation coefficients were 0.96 (20% FB, P ≤ 0.001) and 0.89 (80% FB, P ≤ 0.01). There was no significant correlation between forcing GDH°C accumulated at 20°C and flowering dates at 20% FB (r=0.37, non-significant at P=0.05). Early flowering cultivars, 'Okinawa', 'Yaguchi' and 'Nakatejiro' needed less CU and GDH°C, whereas intermediate cultivars ('Shiroshidare', Hokimomo, 'Zansetsushidare' and 'Genpeishidare'), and late cultivars, 'Sagamishidare' and 'Kikumomo' needed more CU and GDFPC; however, intermediate cultivars such as 'Kyosarasa' and 'Kanpaku' required less CU but more GDH°C. These results suggest that CU for breaking dormancy, response to prolonged CU up to 350, and GDH°C determine the flowering periods of ornamental peaches.
A Japanese pear 'Kumoi' was previously determined as S3S4 by pollination tests, but its S-genotype was reconsidered following our PCR-RFLP (S1 to S9) analyses and pollination tests. Based on its compatibility with 'Seigyoku' (S3S4), and PCR-RFLP analysis, 'Kumoi' was classified as S1S3 for the first time. Additional pollination tests were necessary to prove our contention, but 'Kumoi' did not supply sufficient flowers. 'Sekaiichi' was also assigned as S1S3 by PCR-RFLP analysis, and incompatibility with 'Kumoi'. Instead of 'Kumoi', 'Sekaiichi' was pollinated with the pollen from an 55-homozygote and that from an Ssm4-homozygote. The lack of fruit set revealed that 'Sekaiichi' was incompatible with the S3 and Ssm4 pollen, leading us to predict that the 5-genotype of 'Sekaiichi' was S1S3 or 8384. Two S-genotypes with S1S3 and S2S3 segregated in hybrid progenies between 'Doitsu' (S1S2) and 'Sekaiichi', indicating that S1 was present in 'Sekaiichi'. These results of pollination tests with 'Sekaiichi' indicated the S-genotype of 'Kumoi' was S1S3.
An experiment was carried out in a satsuma mandarin orchard with a bahiagrass (Paspalum notatum Fliigge.) covercrop to evaluate if the concentration of eupalitin, a growth stimulatory compound for arbuscular mycorrhizal (AM) fungi, is correlated with AM fungal development in soils. Root and soil samples were obtained monthly from the satsuma mandarin and bahiagrass to evaluate the AM infection in the roots and the number of AM spores in the soil. The root samples were kept in a freezer until analysed for eupalitin. Eupalitin was isolated and purified from bahiagrass root extracts by flash chromatography, gel filtration, and high pressure liquid chromatography (HPLC) and its concentration in each sample was determined based on a standard solution. The percentage of AM infection in both bahiagrass and satsuma mandarin roots was around 80% during the summer but decreased from October. The number of spores started to increase in March, attaining a maximum of around 800 in 25 g soil in June; the count subsequently decreased. Eupalitin concentration in bahiagrass roots was high from May to November, peaking in May and July. The concentration of eupalitin was significantly correlated with the AM infection in bahiagrass and the number of spores in soil, but not for the AM infection in satsuma mandarin. It suggests that there might be an interaction between AM fungal development and growth stimulatory compounds in soil.
Changes in cell wall polysaccharides and physical properties, associated with the ripening of the peach (Prunus persica Batsch) fruit, were characterized. Enzymically inactivated cell walls were prepared from mesocarps of peach fruit harvested periodically. Pectin-associated and hemicellulose- associated polysaccharides were extracted from the cell walls sequentially, and neutral sugar and uronic acid contents in each fraction were determined. Each fraction was then analyzed for its neutral sugar composition by gas-liquid chromatography (GLC). The fractionated polysaccharides were applied to a column of Sepharose CL-4B gel (1.5 × 40 cm) to estimate the molecular weight distribution. The amount of the uronic acid content in water fraction increased dramatically at the over-ripe stage, whereas xyloglucan, a dominant constituent of hemicellulose, gradually degraded throughout the fruit softening period. These data suggest that peach fruit softening involves the following two processes: initially, xyloglucan degrades during which the pectin remains insoluble; secondly pectin becomes soluble concurrently with continues to xyloglucan degradation. These two processes of maturation correspond to the changes of two parameters on the physical properties, determined by the conventional stress relaxation method. Moreover we determined the degree of methyl esterification (DOM) of the pectin in the crude cell wall extract to investigate its property. Based on our results, we discuss the structure of the cross-linkages among the cell wall polymers and those enzymes involved in fruit softening.
The alkali-soluble polysaccharides in the cell wall extracted from immature and over-ripe peach fruit (Prunus persica Batsch) were analyzed. After removing water and the chelate compound (CDTA) soluble polysaccharides from the crude cell wall in the peach fruit tissue, the residual alkali soluble polysaccharides were extracted sequentially with 50 mM Na2CO3, 1 M KOH, and 4 M KOH. Using an ion exchange chromatography (DEAE-Sepharose 2.5 cm × 30 cm), the 1 M and 4 M KOH fractions were separated into three (1 M KOH a-c) and four fractions (4 M KOH a-d), respectively, but Na2CO3 soluble polysaccharides were not fractionated by this method. The acidic polysaccharide in 1 M KOH fraction (1 M KOH-b and c) was subjected to further analysis. The major sugars in fraction 1 M KOH-b and 1 M KOH-c were xylose and arabinose, respectively. The result of gel filtration (Sepharose CL-4B 1.5 × 40 cm) showed that the average molecular weight of the fraction 1 M KOH-b was heavier than that of the fraction 1 M KOH-c; moreso in immature fruit harvested on Aug. 7 than that in over-ripe fruit harvested on Aug. 24. The results of xylanase (EC 22.214.171.124) treatment of fraction 1 M KOH-b and sugar composition analysis using GLC showed that this fraction was composed of complex polysac-charides of xylan and rhamno-galacturonan. Based on the data of acid hydrolysis and sugar composition analysis the polysaccharides in fraction 1 M KOH-c consisted of xyloglucan and rhamno-galacturonan.
The ureides, allantoin and allantoic acid, are believed to play an important role in the storage and translocation of nitrogen in higher plants. Arginine is a major component in the free amino acid pool in some plants. To investigate the behavior of ureides and free amino acids, especially arginine, in developing Dioscorea opposita plants 'Tsukuneimo', their contents as well as those of water and nitrogen in leaves, stems and tubers were quantitatively analyzed. The content of allantoin in the leaves decreased after any fertilization, while that in the stems did not increase after the 1st fertilization, 96 days after planting; it significantly increased after the 2nd fertilization, 26 days later. Allantoin content in tubers remained constant during tuber development. Arginine content in leaves and stems during the growth of 'Tsukuneimo' were lower than 0.15 and 4.8 μmol·g-1FW, respectively. The time course of arginine in stems content was similar to that of allantoin. In tubers, arginine content at 170 days after planting increased to reach 14 times that at 142 days but then decreased, demonstrating that D. opposita transiently accumulates arginine in stems as well as allantoin in leaves and stems but only accumulates the latter in developing tubers.
Tobamoviruses are classified into four pathotypes, P0, P1, P1, 2 and P1, 2, 3, based on the infection patterns in Capsicum plants that carry different L genes, L1-L4. Paprika mild mottle virus Japanese strain (PaMMV-J), a PI pathotype of Tobamovirus, systemically infected L1-homozygote Capsicum plants and induced mosaic on upper uninoculated leaves. The same virus induced systemic necrosis in 19 commercial cultivars and three breeding fixed lines of Capsicum plants resistant and susceptible to the P0 and P1, 2 pathotypes of Tobamovirus, respectively. Genetic analysis showed that resistance to the P0 pathotype of Tobamovirus of these cultivars and fixed lines differed from L1 and depended on a locus, which may be located on or near the L gene locus. Therefore, we designate the new gene as L1a. Both L1a- homozygote and - heterozygote plants showed resistance to the P0 pathotype of Tobamovirus when incubated at 24°C to 30°C, suggesting that resistance of the Lla to P0 pathotype of Tobamovirus is temperature- and gene dosage-independent. Interestingly, L1a-homozygote but not -heterozygote plants are resistant to PaMMV-J when incubated at 24°C but not at 26°C, indicating that L1a plants resistant to PaMMV-J are temperature- and gene dosage-dependent.
Melon accessions were screened for resistance to Melon necrotic spot virus (MNSV) after mechanical inoculation to determine the virus inheritance pattern. Of the 802 melon (Cucumis melo L.) accessions inoculated with MNSV-NH, 45 did not develop necrotic lesions on inoculated leaves nor did the enzyme linked immunosorbent assay (ELISA) test positive for the virus. These accessions were classified as resistant to MNSV because they also did not develop necrotic lesions after inoculation with two other MNSV isolates. F1 progenies, derived by crossing the resistant accessions and a susceptible 'Natsukei 6', developed necrotic lesions on leaves but the F1 progenies resulting from crosses between resistant accessions and the resistant Terlita' did not. These results suggest that the MNSV resistance of these accessions is recessive and located at the same locus as that in 'Perlita'.
Chrysanthemum (Dendranthema grandiflora (Ramat.) Kitam.) plants, sprayed with a 25 ppm uniconazole one month after planting, evolved ethylene immediately and attained the highest yield on the third day. Plants that were brushed (mechanical stress, MS) for 30 days produced more ethylene on the third day than did uniconazole-sprayed plants and maintained their high production. Plants treated with aminoethoxyvinylglycine (AVG) yielded the lowest levels of ethylene and displayed no peak. Application of AVG to uniconazole-sprayed or brushed plants decreased ethylene evolution. Gibberellic acid (GA)-like substances in the control plants separated into 4 bands on silica gel TLC plates; authentic GA1 GA3, and GA7 were detected at the same Rf in the control plants. GA1- and GA3-like substance contents decreased markedly in plants sprayed with uniconazole or brushed. The application of AVG alone did not affect GA- like substance content, nor did it enhance GA activities in uniconazole-sprayed plants. Thus, growth of chrysanthemum plants is controlled not only by GA activities but also by ethylene.
We investigated the utilization of arabino-oligosaccharides derived from arabinan, which is abundant in side chains of apple pectin, by intestinal bacteria in vitro to clarify the mechanisms of the beneficial effects of apple ingestion on intestinal bacterial flora. Among the test strains, arabino-oligosaccharides that contain more than three units were selectively utilized by Bifidobacterium adolescentis, Bi. longum, and Bacteroides vulgatus. The digestion of arabino-oligosaccharides by artificial digestive juices was investigated in vitro. Arabino-oligosaccharides were not digested by artificial saliva, artificial gastric juice, or artificial pancreatic juice, and only slightly by small intestinal enzymes. These results suggest that arabino-oligosaccharides serve as prebiotics and that the beneficial effects of apple ingestion on intestinal bacterial flora are partly due to apple pectin.
The medium composition for the production of sporophytes from gametophytes by sexual reproduction in Adiantum capillus-veneris was investigated. The addition of 3% sucrose to Murashige and Skoog's medium (MS medium) significantly enhanced the growth of gametophyte. However, sucrose did not induce the production of a sporophyte. The elimination of NH4NO3 from the culture medium induced the production of sporophytes, although elimination of KNO3 was not effective. Reduction of total nitrogen to 25% of the original MS medium effectively promoted sporophyte yield, indicating that the nitrogenous component is one of the most important factors for the production of sporophytes by sexual reproduction in Adiantum capillus-veneris.
Nine Cattleya species and their allied genera and 36 species of Cattleya were examined to clarify phylogenetic relationships within Cattleyae and Cattleya by using RAPD analyses. Fifteen random primers yielded 70 RAPD markers from 15 species of Cattleya and their allied genera. UPGMA (unweighted pair-group method using arithmetic averages) calculation presented three clusters, among genera with four pollina which indicate that Cattleya was close to Epidendrum secundum, but distantly related to Encyclia cordigera. Among the eight pollinia genera, 4 genera, namely, Brassavola, Laelia, Rhyncholaelia, and Sophronitis, formed one cluster. Cluster analysis shows that Sophronitella is close to Leptotes bicolor having six pollinia. Ninety three RAPD markers obtained from 36 Cattleya species by using 9 random primers presented clusters similar to the morphological classification of Cattleya species proposed by Brieger et al. who recognized 4 subgenera, namely, Cattleya, Diphyllae, Skinneri, and Rhisanthemum. In the Diphyllae section, Guttatae was further separated into two clusters, C. guttata and C. schilleriana. C. kerrii ranked between subgenera Cattleya and Diphyllae. Therefore, further studies on genetic information of species belonging to subgenus Diphyllae are needed.
Water soluble pectin in peach (Prunus persica (L.) Batsch) fruit, which is well known to increase with fruit maturation, was isolated and characterized. The water soluble polysaccharides were extracted from crude cell walls of immature and over-ripe fruits and separated on an ion exchange column (DEAE-Agarose 2.5 × 30 cm). By eluting with 50 mM phosphate buffer (pH 6.0) and then the buffer containing 0.1 and 0.5 M NaCl, the water soluble polysaccharides from unripe and ripe fruits were separated into fraction I and II, and fractions I, II and III, respectively. The polysaccharides in fractions II and III (pectic polymers) were applied to the gel filtration column (Sepharose CL-4B 1.5 × 40 cm) to determine their molecular weights. The data reveal that the molecular weight of fraction III was heavier than that of II. The sugar composition analysis, using gas liquid chromatography, indicated that the two dominant neutral sugars in fraction II were galactose (30-40%) and arabinose (40-48%), whereas in fraction III, it was arabinose (more than 65%). These data show that fractions II and III have different neutral side chains of pectic polysaccharides.