Flowering in Radish

The relationship among gibberellins, C vernalization, and photoperiod in the flowering response of radish, Raphanus sativus L., cv. Miyashige-sofuto, was studied. The optimal condition for flowering was vernalization and a 16-hour photoperiod; GA3 had no additional effect. Gibberellin A3 (60 ,jg total) was not able to induce flowering in nonvernalized plants grown on 8-hour days, but it did increase the percentage of nonvernalized plants that flowered under long days from 60 to 100. Gibberellin content of vernalized seedlings increased within the first 24 hours after seedlings were transferred to the greenhouse. Content reached a peak in the first 4 days after transfer and thereafter remained constant. Essentially no gibberellin was found in 2 day-old nonvernalized (control) seedlings of comparable size to the vernalized ones. Gibberellin content in the controls reached a peak on the fourth day of growth in the greenhouse; thereafter, it decreased steadily. Bolting was inhibited slightly by CCC when applied during vernalization; it was almost completely inhibited when CCC was applied after seed vernalization. Extraction experiments revealed that CCC actually reduced the gibberellin content when applied during or after vernalization. The dwarfing agent, however, had essentially no effect on flowering. We concluded that gibberellins likely play a direot role in bolting of 'Miyashige-sofuto' radish, but probably are not directly functional in initiating flowering. Gibberellins induce bolting and flowering under noninductive conditions in many plants that require low temperature for flower initiation (7). Wittwer and Bukovac (14) showed that application of GA3 to radish plants accelerated flowering under both long and short days. Kagawa (5) reported that flowering of nonvernalized radish was induced by GA3 in the fall or spring, whereas GA3 only promoted bolting in the summler. He attributed flower initiation in the fall and spring to a combination of gibberellin and low tenmperature. The role of endogenous gibberellins in the regulation of stem elongatioIn and flower formation in radish has not been studied, although Murakami (10) was unable to detect gibberellin-like activity in mlature dry seeds of a Japanese radish cultivar. Dwarfing agents, such as 2-chloroethyltrimethyl amnmonium chloride (CCC) and (4-hvdroxy-5-isopropyl-2-methylphenyl) trimethyl ammonium chloride-i-piperidine carboxvlate (AMO-1618), which inhibit gibberellin production, have been useful in studying the role of endogenous gibberellins in flowering of a long-day plant (2), of a .short-day plant (15), of a long-short-dav plant (17), and of plants requiring low temperature for flower formation (8, 13). In these instances tlle growtlh retardlants A10-1618 and CCC apparently reduiced I On leave from Natiolnal Ilnstitute of Agricultural Sciences, Kita-kii, Tokyo,Japan, the level of endogenous gibberellins below that required for flowering. The inhibition was overcome by GA3. Direct analysis of endogenous gibberellins has provided evidence indicating a role for gibberellins in the flowering process of some cold-requiring and long-day plants (1, 4, 6, 11, 13, 16). The results indicate that endogenous gibberellins change qualitatively or quantitatively or both in relation to initiation of flowering. II this study an effort was made to identify the role of gibberellin in the bolting anid flowerinig processes of radish. Evidence wvill be presented to support the view that gibberellin plays a primary role in inducing bolting and not flowering, at least in the cultivar under study. Materials and Methods The Miyashige-sofuto cultivar of radish, Raphialitns sativits, was used as test material in all experiments. This cultivar is popular as food in Japan. Effects of Gibberellins on Nonvernalized Planits. The effects of gibberellins A,, A3, and A7 (containiiig 4 % GA4) on the floweriing response of radish plaints wvere studied. Seeds were germinated in petri dishes at room temiiperature for 3 days. Then the seedlinigs were transferred to plastic pots in a greenhouse on September 11. Each treatment had 7 replications of 2 plants each. Application of the gibberellins was started whlien the planits lhad 1208 www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 1968 American Society of Plant Biologists. All rights reserved. SUGE AND RAPPAPORT-ROLE OF GIBBERELLINS IN STEM ELONGATION AND FLOWERING 1209 5 leaves, 15 days after germination. Fifty PI of each gibberellin was applied to the apex of the plants in each respective treatment. This was done twice weekly for 10 weeks. Effects of Vernalization, Photoperiod, CCC, and Gibberellinis on Stern Elongation and Flowering. Seeds were germinated 2 days at room temperature in water contained in petri dishes. The seedlings were then transferred to other dishes containing water or 10-2 \i CCC, and vernalized 20 days at 5'. Nonvernalized (control) seeds were soaked at room temperature in dishes for 2 days just prior to the end of vernalization, thus providing seedlings of approximately the same size and appearance as the vernalized ones. Both lots of seeds were planted in plastic containers, 1 plant per pot, and grown in chambers under 8or 16-hour photoperiods. The light source consisted of twelve 300-watt incandescent lamps set 2 m above the plants. Temperature was controlled at 25' during the light period and 20' during the dark period. Five days after transfer both the vernalized and nonvernalized plants were divided into 3 groups of 10 plants each. Then GA3 (0, 1, or 10 ug/plant) was applied to the apex of each plant every 3 days in 50 ul of a solution prepared in 0.05 % Tween-20 (polyoxyethylenesorbitan monolaurate). Total amounts applied are shown in table II. For comparison, GA7 (10 ug/plant) was applied to one lot of 10 nonvernalized plants grown under an 8-hour photoperiod. To test the effects of CCC applied after vernalization, 4 nonvernalized seedlings (cotyledons expanded) wN-ere sown in each of 15 plastic pots in a room controlled at 5°. After 20 days of chilling, the planits wsere transferred to growth chambers controlled at 25' during the 16-hour light period and at 20' in the 8-hour dark period. Twenty milliliters of a solution containing 5900 mg/liter CCC were applied to the soil of each pot at 0, 3, and 6 days after the end of the vernalization treatment. The treated pots each received a total of 354 mg of CCC. To test its ability to overcome retardation, GA3 (20 jug/plant) was applied to the apex of the CCC-treated plants in 5 pots. Twenty plants were used per treatment. Dates of bolting, flower bud appearance and anthesis, and stem heiglht and number of leaves per plant at anthesis were recorded in all growth experiments. Effects of Vernalization and CCC on Content of Gibberellin-like Substances. Planit Materials. Vernalized and nonvernalized seedlings wvere planted in vermiculite in plastic containers and grown under continuous light in a greenhouse. Additional light was provided bv 4 20-watt fluorescent lamps. Plants were extracted immediately or 4, 10, or 23 days after the seedlings w-ere transferred to the greenhouse. The appearance of the plants was as follows: Day 0, cotyledons partly expanded; no apparent difference between vernalized and nonvernalized plants. Day 4, cotyledons fully expanded; no apparelnt difference betweeni vernalized and nonvernalized plants. Day 10, seedstalk formation in vernalized plants. Day 23, flower buds visible in vernalized plants. The effect of CCC on the production of gibberellin-like substances in vernalized radish plants was studied using 12-day-old seedlings kept at 5° for 20 days. Two grams CCC were added to half the pots at the start of vernalization, and to the remainder when vernalization was completed. The plants were transferred to a greenhouse and grown under 24-hour photoperiod for an additional 10 days. Extraction and Fractionation. The plants (minus the roots) were covered with 70 % acetone and ground in a blender. After the homogenate was shaken for 8 hours, it was filtered through cheesecloth and filter paper. The filtrate was evaporated to the water phase under reduced pressure, adjtusted to pH 2.0 with phosphoric acid, and extracted 3 times with ethyl acetate. The combined ethvl acetate fraction was further extracted with phosphate buffer at pH 7.0. The buffer, adjusted to pH 2.0 with phosphoric acid, was extracted 5 times with ethyl acetate. Following overnight dehydration of the combined fraction with anhydrous sodium sulfate, the fraction was concentrated under reduced pressure. Thin Layer Chromtatography. Each concentrated extract was applied, in a small volume of acetone, as a 0.5-cm band to a 20 X 20 cm thin-layer plate prepared with 0.2 mm thick silica gel. The solvent, isopropanol-ammonium hydroxide-water (10 :1:1 v/v), was permitted to run a distance of 10 cm on the plates. After drying, the chromatogram was divided into 10 equal zones. and the silica gel scraped off into test tubes each containing 0.5 ml water. Bioassay. 'Tan-ginbozu' dwarf rice seedlings were used as test plants. Five seedlings (sshoots 1 mm in length) were placed in each test tube coIntaining the silica gel, and were growni 5 days at 30' unider 24-hour photoperiod (light intensity about 4000 lux). Length of the second leaf sheath of the seedlings was measured 5 days later. Gibberellin content was expressed as GA3 equivalents per 100 g fresh weight of tissue. The amount was calculated using a standard curve obtained fronm the response of rice seedlings to GA3.