Anther culture of‘Trovita’orange, a cultivar of sweet orange (Citrus sinensis Osbeck), was attempted. Anthers with microspores at uninucleate stage were cultured on the Murashige and Skoog′s medium(9) supplemented with or without indole-3-acetic acid (IAA) and kinetin at 28°C in darkness. Embryoids appeared directly from within anthers after about 10 weeks of culture. The media containing kinetin and IAA both at 0.002 or 0.02mg/l were effective in embryoid formation. In addition, 0.2 or 2.0mg/l kinetin and 0.2mg/l IAA seemed to be also favorable when combined with 0.002 or 0.02mg/l IAA and 0.002 or 0.02mg/l kinetin, respectively. Calli were formed well on the media with kinetin at 2.0mg/l followed by those with IAA at 2.0mg/l. After incubation in light with a 16-hour photoperiod, some embryoids differentiated shoots. Induction of roots was stimulated by transfer of embryoids from the embryoid induction media containing growth regulators to a medium without them. Some root tip cells of differentiated plantlets had a diploid chromosome number of 18.
Some experiments were carried out to obtain further informations concerning the effect of bud pollination on overcoming the self-incompatibility of Hyuganatsu, Citrustamurana Hort. ex Tanaka. The results obtained are as follows. 1. By means of chromatofocusing analysis, physiological differences between mature and immature flower buds were clarified with respect to their proteinous components in pistils. As to soluble proteins, glyco-proteins and peroxidase isozymes in stigmas, styles and ovaries, there existed clear differences between mature and immature flower buds. But, no remakable differences on esterase and alkaline phosphatase isozymes were observed between the two kinds of flower buds. 2. Scanning electron microscopic observation revealed that stigma exudate of immature flower buds was less abundant than that of mature ones. It also showed that covering of Hyuganatsu pollens by exudate on stigmas of immature flower buds was not so rapid as that on the stigmas of mature flower buds. According to fluorescent microscopic observation, the pollen germination on the stigmas was rather delayed in immature flower buds as compared with mature ones. Nevertheless, the pollen tubes grew smoothly through the styles of immature flower buds, and entered ovaries 7 days after pollination.
Amino acids, amides and nitrate were determined in the xylem sap obtained by the vacuum extraction from sections of 2 to 3-year-old wood of 21-year-old satsuma mandarin trees (Citrus unshiu Marc.), and seasonal variations in their content were followed throughout the year. The main nitrogenous compounds identified were proline, arginine, asparagine and nitrate, all of which showed seasonal variations in their content. Proline showed a slight increase in April, the early stage of new shoot development, then a sharp decrease until early June, and again an increase from October to December. Arginine showed a similar seasonal variation to that of proline. Asparagine showed 2 peaks, one in mid May, the other in late July. Nitrate had a similar variation to that of asparagine. Other amino acids showed smaller variations compared with the above-mentioned compounds.
Buds, leaves and 2 to 3-year-old bark and wood were sampled from 21-year-old satsuma mandarin trees (Citrus unshiu Marc.) throughout the year and analysed for total nitrogen, 70% ethyl alcohol soluble and insoluble N, amino acids, mainly arginine and proline, and amides. Many nitrogenous components showed seasonal variations in their content. In old parts, it was the highest in April, the sprouting season, and the lowest in July, the final stage of new shoot development, while it was lowest in September in new leaves. Bark and wood showed temporary increases in the content of total and soluble N, free proline, and especially free arginine during the sprouting period from late March to mid April, and then declines in insoluble N. Buds, on the other hand, showed a marked decrease in proline together with a drastic increase in arginine during the sprouting period. These findings suggest that the soluble N components, such as proline and arginine, are used first for new shoot development, followed by the insoluble N. In old leaves, most of these components began to decrease in early May and the percentage decline of total N from early May to July was about 16% on a dry weight basis, about 40% of which was accounted for by the loss of free proline N. On the whole, all of these components increased in all parts of the tree from autumn to winter. The increases of proline in bark and leaves were particularly marked.
In 14 to 16-year-old‘Campbell Early’grapevines, 5-chloroindazol-8-acetic acid (ethychlozate) and naphthaleneacetic acid (NAA) increased flower abscission 5 to 8 days after they were applied to the leaves on the shoot each at 50 to 200ppm at full bloom, although such effect varied with seasons considerably. Moreover, they caused wilting of the shoot tip and leaves beginning several hours after the treatment, from which the shoot recovered after 7 to 10 days. When ethychlozate and NAA were applied to the flower cluster at full bloom at 100 and 200ppm, they increased not only the flower abscission but also the set of small seedless berries (shot berries). Ethychlozate and NAA had no effect on the sugar content of the must, while tended to decrease the total acidity especially at high concentrations. Transport of photosynthates in the vines was studied by feeding given leaves with 14CO2 1, 4 and 7 days after NAA application using 3-year-old potted vines. The total 14C exported from the fed leaf were markedly less in the vines with foliage application of NAA (foliage-treated vines) than with cluster application (cluster-treated vines) and without NAA application (control vines). 14C assimilated by theleaf 2 nodes above the flower cluster was transported mostly to the shoot tip and stem above the cluster (upper parts) and little to the trunk and stem below the cluster (lower parts) and the cluster in the control and cluster-treated vines. While, in the foliage-treated vines, a fairly high proportion of 14C was found in the lower parts. After feeding the leaf just above the flower cluster, some proportion of 14C was transported to the cluster in all treatments. In the control vines, transport to the upper parts decreased while that to the cluster increased as the feeding time was delayed to 7 days after the treatment. In the cluster-treated vines, transport to the upper parts decreased, while those to the lower parts and flower cluster increased markedly after feeding 7 days after the treatment. On the contrary, in the foliage-treated vines, transport to the upper parts was small after earlier feedings, while after feeding 7 days after the treatment it increased markedly accompanied with a sudden decrease in that to the flower cluster. From these results, transport of the photosynthates to the flower cluster was assumed to be involved in the berry abscission following NAA application.
Volume estimation was made on a cucumber fruit as attached to vine.‘Fruit volume index (FVI)’was calculated by FVI=l×(a12+a22+a32), where l is the fruit length and a1, a2 and a3 are the respective girths at 3 positions along the fruit length. FVI had high correlations with volume (r>0.999), fresh weight (r>0.999) and dry weight (r>0.99) of a fruit. FVI gave estimation of the volume, fresh weight and dry weight of a fruit with errors of 3%, 3% and 0.2g, respectively. This method enabled us to determine the growth rate of a fruit even at intervals as short as 4 hours with sufficient accuracy. The growth curve of a cucumber fruit was single sigmoidal. A Gompertz curve, which was obtained by using data at the growth stages from 1 to 80% final fruit volume, fitted well over the whole growth period for cv.‘Tokiwa Hikari 3 Go A Gata’, but not for cv.‘Kurume Ochiai H Gata’. For neither cultivar, a logistic curve showed better fitness to the whole period of fruit growth than a Gompertz curve did. RGR of a fruit was highest when it was 9 to 20cm long, followed by a rapid decrease. RGR of a fruit with the length shorter than 20cm showed clearly a diurnal change with a maximum value between 12:00 and 20:00. Daily means of RGR were higher on fine days than on cloudy days. RGR was higher in spring than in autumn. Fruits of cvs.‘Tokiwa Hikari 3 Go A Gata’and‘Kurume Ochiai H Gata’were different in their size at the matured stage as well as in their time to reach that stage. However, they were not different in RGR before they reached 20cm long.
Germination of the spinach (Spinacia oleracea L. var. grabra cv. Nobel) seed was examined at supra-optimal temperatures. Almost all seeds germinated rapidly at 20°C. However, germination decreased as temperature was raised and fell to about 10% at 35°C. Removal of the pericarp as well as presoaking treatment markedly improved the germination, especially, at 35°C. Germination of pericarp-removed seeds was inhibited by water soluble extracts from seeds and pericarp, but not by those from presoaked seeds. The inhibitory activity of the extracts was not affected much by the incubation temperatures before extraction. Sulfuric acid treatment conducted to modify the physical properties of the pericarp and hydrogen peroxide treatment conducted to elevate the oxygen tension promoted germination. Thus, the poor germination at high temperatures may be caused mainly by the pericarp, which produces germination inhibitors or gives mechanical resistance and impermeability to gases. Even in the pericarp-removed seeds, germination was delayed at high temperatures, indicating that high temperature directly affected the embryo.
The present studies were designed to elucidate the effects of application of growth regulators on the low temperature induction of flower buds in onion plants cv. ‘Sapporo-ki’. 1. Plants were treated with daminozide (SADH) at 2, 000ppm, with ethephon at 500 and 1, 000ppm and with gibberellin (GA3) at 100ppm before low temperature (9°C) exposure. Both the plant growth and induction of flower buds were suppressed a little by SADH, while strongly by ethephon. On the contrary, GA3 increased the number of leaves and hastened the induction of flower buds. 2. In the plants with leaf sheath diameter of 10mm, the minimum duration of low temperature exposure required for the induction of flower buds was about 20 days and little affected by GA3, but in the plants with leaf sheath diameter about 7mm, it was 40-50 days and shortened to about 20 days by GA3. 3. GA3 was effective in the induction of flower buds when it was applied before or during the low temperature exposure, but it was ineffective when applied after the low temperature exposure. It seems that GA3 hastens the flower bud formation through its effect on the activation of meristematic tissues and not through taking the place of low temperature in its effect.
This study represents the first case of getting orchid seedlings directly from ovules in vitro culture of ovaries or ovules excised before fertilization. By the usual invitro culture of mature seeds, at least a period of 240 days was required from pollination to obtaining seedlings of D. pulcherrima. By the new method with pollinated but not fertilized ovaries or ovules, the period could be shortened by about 150 days. Moreover, the seedlings obtained proved to be diploid, suggesting the occurrence of fertilization during the culturing. In ovule culture, seedlings were obtained only when the basal medium was supplemented with sucrose or coconut water which seemed to supply sucrose in some form of carbohydrates. While in ovary culture, they were obtained without the addition of sucrose, perhaps because sucrose was supplied from the ovary wall.
To promote the development of bulblets formed from leaf segments of Lilium rubellum Baker, the bulblets were excised and subcultured in a modified Murashige and Skoog medium (12) (a basal medium) supplemented with different concentrations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA), alone or in combination, both in diffused light at 150-300lx and in continuous darkness. In addition, the leaf emergence from the bulbs obtained by subculture was examined in the field. 1.NAA was the major factor in increasing fresh weight and root numbers of excised-bulbs. The optimum concentration was 0.1mg/l when NAA alone was added to the basal medium. 2. Effects of BA on the development of excised-bulbs varied with the concentrations of NAA added to the basal medium. BA at concentrations of 0.001mg/l synergistically increased the fresh weight of the excised-bulbs. In addition, excised-bulbs formed new adventitious bulblets when they were cultured in the basal medium supplemented with more than 0.01mg/l NAA and less than 0.1mg/l BA. However, the addition of 0.1mg/l BA in combination with NAA at any concentration added to the basal medium repressed the development of excised-bulbs and the formation of bublets. 3. When the excised-bulbs were cultured in diffused light in the medium containing 1mg/l NAA and 0.1mg/l BA, the highest increase in fresh weight of the bulbs occurred, but these concentrations of the growth regulators caused the aberrant growth of the bulbs. Thus, the bulbscales scarcely grew and the basal part of the bulbs (disc) enlarged abnormally. 4. Rooting of excised-bulbs was enhanced by the addition of only NAA at concentrations of 0.1 or 1mg/l to the basal medium. However, BA at any concentration lessened the rooting of excised-bulbs and at 0.1mg/l, almost completely inhibited rooting when combined with NAA at any concentration. 5. Increases in fresh weight, rooting and adventitious bulblets were generally superior in the excised-bulbs which had been cultured in diffused light when compared to those cultured in darkness. 6. The leaf emergence in the field was more excellent in the bulbs subcultured in diffused light than in the bulbs subcultured in darkness. NAA and BA supplemented in the subculture had scarcely any effect on the leaf emergence from the bulbs in the field. In some cases the addition of NAA and BA suppressed the leaf emergence, and the supression was quite noticeable in the bulbs subcultured in darkness in medium including 0.1mg/l BA and NAA at any concentration. It can be concluded from these results that the extreme development of bulbs excised from leaf segments of L. rubellum Baker was performed by subculturing the excised-bulbs in diffused light in a modified Murashige and Skoog medium supplemented with 0.1mg/l NAA and 0.001mg/l BA.
Ripening characteristics of‘Cavendish’bananas were investigated, especially on the plant at plantation in the Philippines and off the plant after transport to Japan. (This strain should be accurately referred to‘Giant Cavendish’, belonging to Musa AAA group, Cavendish subgroup, and have been occupied the majority of banana trade in Japan.) Commercial bananas were ripened at temperature of 15, 20, 25, 30 and 35°C without ethylene treatment, or at 20°C with ethylene treatment at various concentrations of 1-5000ppm. Temperature of 30°C and above inhibited the degreening of skin and particularly 35°C produced fruit known as“boiled”or“cooked”. Normal ripening was observed at 15°C except the disappearance of green tips even at the final stage of ripeness. Temperatures ranging from 20 to 25°C seemed to be optimal far the ripening of‘Cavendish’fruit. No difference in any aspect of ripening was found among fruits treated with various concentrations of ethylene. ‘Cavendish’bananas at the plantation in the Philippines did not ripen enough for eating while attached on the plant, but finally became yellow with splitting approximately 120 days after flowering. Little changes in carbon dioxide production and in sugar content with very low levels were observed, and also no evolution of ethylene was found during the development on the plant until splitting occurred. About 90 days after flowering, however, physiological ripening seemed likely to start from the findings that organic acid sharply increased coinciding with decreasing of starch. Fruits were harvested 65, 75, 85, 95 and 105 days after flowering, and ripened at 25°C after transport to Japan. Bananas for 65 or 75 days had more than 10 days of green-life, while those for 85 or 95 days had less than 4 days of it. Fruits harvested at 105 days lost their green-life on the boat with yellowing. In any ripening aspect after onset of climacteric, however, little difference was found
The experiment was carried out to clarify the physiology of the fruit ripening regulated by ripening inhibitor gene, rin, of tomato (Lycopersicon esculentum Mill.). Changes in respiration, ethylene production, and ABA, IAA, soluble carbohydorate, titratable acid and free amino acid contents during ripening were investigated in detached fruits of tomato cv.‘Rutgers’and its nearly isogenic rin mutant stock. 1. Maximum production rates of carbon dioxide and ethylene production from ‘Rutgers’fruit were observed during storage but rin fruit showed little change in the production rates of carbon dioxide evolution and low rates of ethylene production. 2. Free ABA content in pericarp tissue during storage gradually increased and reached peak levels 9 days after harvest (dark pink stage) in both‘Rutgers’and rin fruits. Its content was higher in rin rather than in‘Rutgers’fruits. Bound ABA content changed in parallel with free ABA content in pericarp tissues of both ‘Rutgers’and rin although the former was always lower than the latter. 3. IAA in pericarp tissue increased during storage and reached a maximum level at dark pink stage of ripeness in‘Rutgers’fruit but, in rin fruit, only traces of it were observed. 4. Soluble carbohydorate and titratable acid content in pericarp tissue decreased during storage and no differences were observed between‘Rutgers’and rin fruits. 5. The predominant free amino acids in pericarp tissue during storage were aspartic acid, threonine+serine fraction, glutamic acid, phenylalanine and γ-amino-butyric acid. Contents of aspartic acid and glutamic acid progressively increased during storage in both‘Rutgers’and rin. In other amino acids, little changes were observed.
Chinese chive is in demand as a seasoning vegetable all the year round. But, the deteriorations such as yellowing, rotting and wilting are very rapid at high temperatures. Thus, the shelf life is only one or two days in summer season, which is becoming a serious problem. Prevention of the deterioration by precooling combined with the seal-packaging with polyethylene bag was studied in this experiment. Precooling combined with the seal-packaging was very effective in preventing yellowing, rotting and wilting as well as in reducing respiration and losses of some components such as chlorophyll, reduced ascorbic acid and carotene. The seal-packaging alone was fairly effective in preventing the deterioration, but at high temperatures, it caused the metabolic abnormality resulting in visible injuries and abnormal odors. In such cases, making a few pin-holes in polyetylene bag prevented the abnormality, and prolonged the shelf life in some extent. Based on these results, a recommendable shipping system was established and applied practically. It may be summarized as follows. 1. Chinese chives are prepared and bundled with a rubber band by about 110g. 2. Ten bundles are seal-packaged in a polyethylene bag (size: about 30×45cm, film thickness: about 0.025mm, with or without a few pin-holes). 3. Four seal-packaged bags are vertically packed in a corrugated box (size: about 26×30×36cm, with side vents), and precooled to about 5°C by room cooling method within a day. 4. The precooled produce is shipped generally by trucks with insulated box or sheets. In this system, strict adherence to optimums in precooling and transit can be relaxed a little because of modified atomosphere effects by the seal-packaging.