The involvement of photosynthesis in inducing bud formation on excised leaf segments of Heloniopsis orientalis (Liliaceae)

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1 Plant & Cell Physiol. 19(5): (1978) The involvement of photosynthesis in inducing bud formation on excised leaf of Heloniopsis orientalis (Liliaceae) Yukio Kato Biological Laboratory, Fukui University, Fukui 9, Japan (Received January 11, 1978) The role of photosynthesis in inducing adventitious bud formation on leaf of Heloniopsis orientalis was investigated. The effect of white light reached a maximum at about 125 J-m~ 2 -sec~ 1. White, red, blue and far-red light were effective in inducing bud formation, but green light was not. In darkness, bud formation was induced if sugar was added to the nutrient medium. The photosynthetic inhibitors DGMU and AT blocked the effect of light. Bud formation was inhibited in CO2-free air. The requirement of sucrose for bud formation in darkness could be replaced by citrate. It was concluded from these results that light appears to induce buds on leaf through some processes dependent upon photosynthesis. Key words: Adventitious bud formation Heloniopsis (Liliaceae) Photosynthesis. It is well known that the formation of adventitious buds on Bryophyllum tubiflorum (12), B. daigremontianum (2) and BegoniaXcheimantha (1, 3) leaf cuttings is greatly influenced by day length. Heide (4) suggested that these photoperiodic effects are mediated by changes in endogenous cytokinin and auxin contents. However, the contribution of photosynthesis by the treatment of the photoperiodic cycle to the carbohydrate content of leaf remains undetermined. Heloniopsis orientalis easily forms adventitious buds on isolated leaves and excised leaf fragments (8). Growth regulators, especially BA, added to the basal medium stimulated bud formation in light (6"). However, this cytokinin is ineffective in darkness. Bud formation occurs in darkness without exogenous growth regulators if carbohydrates are added to the basal medium (7). Experiments on the influence of pre-incubation in darkness and on successive treatment with sorbitol (a sugar alcohol ineffective on bud formation in darkness) and sucrose (an effective carbohydrate) suggested that photosynthesis is involved in inducing bud formation on excised leaf (7). The present paper describes the results of an attempt to clarify the role of the processes dependent upon photosynthesis in bud formation. Downloaded from at Penn State University (Paterno Lib) on September 16, 16 Materials and methods The preparation and aseptic culture of leaf of Heloniopsis orientalis Abbreviations: BA, 6-benzyladenine; DCMU, 3-(3,4-dichlorophenyl)l,l-dimethylurea; AT, 3- amino-1,2,4-triazole. 791

2 792 Y. Kato Table 1 Some characteristics of leaves and explants used as experimental material Sources of plants Average leaf Average leaf Average leaf Size of fdla theieht-cm') length in cm width in cm thickness in n explants * " 6 ; (Range of variation) (Range of variation) (Range of variation) cultured-cm Young plants (3-5) Green leaves Mature plants (16-18) Green leaves 3.0 ( ) 2.0 ( ) 13.0 ( ) 0.6 ( ) 0.3 ( ) 2.4 ( ) 2 ( ) 0.7x (0-190) 0.3x (290-3) 1.0x1.0 (Thunb.) C. Tanaka were similar to those previously described (6", 7). Some characteristics of the leaves and explants used as experimental materials are shown in Table 1. Leaf from etiolated plants were sometimes used and prepared as follows: The shoot buds regenerated on mature leaf were grown on Knop's agar medium supplemented with Nitsch's trace element solution (8) and 2% sucrose in darkness for about one month. Slender, elongated leaves characteristic of the etiolated plants were excised and were trimmed to 0.3 X 1.0 cm. For the cultivation of excised leaf, the formula of Murashige and Skoog's (9) major and minor mineral elements and vitamin mixture was employed. In some experiments sucrose and organic acids were added to the basal medium. Two metabolic inhibitors, DCMU and AT, were added to the basal medium either singly or in combination to inhibit photosynthesis. Cultures were maintained in a culture room at 23±2 C. A spectroradiometer (YSI Model 64A, Yellow Springs Instrument Co.) was used to measure all light intensities. White light (light intensity, 125 J-m^-sec" 1 ) was supplied by 40-watt cool white fluorescent lamps (Toshiba SW NL). Blue light (130 J-m-^sec" 1 ) was supplied by filtering the white light through a 3-mmthick blue plexiglass plate (Rohm and Haas, No. 45), red light (130J-m- 2 sec~i) through a 3-mm-thick red Matsuda glass-filter (Toshiba V-R2 B) and green light (150 J-m~ 2 -sec~ 1 ) through a 3-mm-thick green Matsuda glass-filter (Toshiba V-Gl). Far-red light (45 J-m^-sec" 1 ) was supplied byfilteringlight from a 0-watt tungsten bulb through a 3-mm-thick red Sekisui Deluglass K (Sekisui No. 2) and a 3-mmthick blue Sekisui Deluglass K (Sekisui No. 306). A -cm-deep filter was placed between the tungsten bulb and the filter to keep a constant temperature. Using a culture flask (0 ml) with a central well sealed with aluminum foil and two layers of Parafilm M (American Can Co.), the influence of the presence of as an absorbent of CO 2 was investigated. The number of with buds and the average number of buds per were determined in each treatment. Downloaded from at Penn State University (Paterno Lib) on September 16, 16 Effect of light intensity Results Fig. 1 shows the effect of the intensity of white light. In a range between 75

3 Role of photosynthesis on bud formation I Fig. 1. Effect of light intensity. Leaf from young green ( ) and etiolated (O) plants were used. The solid line shows the percentage of with bud(s) and the broken line the number of buds per. 0 If^"" ~^ J LIGHT INTENSITY (J.M~ -SEC," 1 ) and 125 jtn~ 2 -sec~ 1, the stronger the light intensity was, the higher the frequency of leaf with buds. At the highest intensity (175 J-m^-sec-i) bud formation was rather inhibited, probably because the present material is a shade plant. Light quality In total darkness bud formation did not occur on leaf from either green (mature and young) or etiolated plants; whereas it occurred under light, irrespective of its quality. The frequency (%) of buds on etiolated leaf under green, blue and far-red light was quite low (Table 2). Segments from mature leaves, however, formed more buds in these light conditions. It is reasonable to assume, therefore, that the effect of these light conditions on bud formation is dependent upon the physiological status before and after excision or upon leaf age. The influence of sucrose on bud formation in from light-grown young leaves was investigated under continuous illumination with red, green, blue, far-red light and darkness. In darkness, buds were produced on the basal medium alone (Table 2), but a higher frequency of with buds was observed on the sucrose-containing medium (Table 3). The frequency of with buds on the basal medium alone under green light was far less than that under other light conditions (Table 2). Segments grown on the sucrose-medium, however, allowed 81.8% bud formation under the same green light (Table 3). Effect ofdcmu To investigate the role of photosynthesis in the light effect, experiments were conducted in which the were grown on the basal medium containing the photosynthetic inhibitor DCMU under all light conditions as described before. This chemical was tested in concentrations from ~ 4 M to ~ 6 M. DCMU at ~ 4 M and ~ 5 M inhibited bud formation on both mature and young leaf (Fig. 2). DCMU at ~ 5 M also inhibited bud formation under all light conditions (Tables 2 and 4). In the green light only 5.3% of the mature leaf formed buds. Higher frequency of bud formation was observed on the mature leaf fe Downloaded from at Penn State University (Paterno Lib) on September 16, 16

4 794 Y. Kato Table 2 Bud formation of leaf grown under continuous illumination of white, blue, green, red and farred light and in darkness ' Light condition White Blue Green Red Far-red Light intensity (J-m-2-sec-i) Source of Number of cultured " Observed 60 days after the start of culture. * Statistically different from the control (white light). with buds (%) (0.0)* (0.0)* (0.0)* (90.0) (0.0) (0.0) (0.0) (73.9) (50.0)* (58. 3)* (28.6)* (30.9)* (0.0) (75.0) (82. 2) (80.0) (80.0) (.3)* Average number of buds per 0.0± ± ± ± ± ± ± ± ±0.05 Average plant height-mm ± ± ± ± ± ± ± ± ± ± ± ± ±0.77 grown under various wavelengths of light, if sucrose was supplied to the DCMUcontaining medium (Table 4). These results imply photosynthetic involvement in the light effect. Table 3 Bud formation in young leaf in the presence of sucrose (2%) under continuous illumination of white, blue, green, red and far-red light and in darkness" Light condition White Blue Green Red Far-red cultured Observed 55 days after the start of culture. with buds 34 (89.5) 15 (83.3) 22 (0.0) 18 (81.8) (0.0) (93.3) Average number of buds per 3.3 ± ± Average plant height-mm 33.4 ± ± ± ±0.46 Downloaded from at Penn State University (Paterno Lib) on September 16, 16

5 Role of photosynthesis on bud formation DAYS AFTER CULTURE DAYS AFTER CULTURE Fig. 2. Effect of DCMU. Leaf from young (solid line) and mature (broken line) plants were used. A, % of with bud(s); B, number of buds per, a, control; b, ~ 6 M; c, -s M; d, -4 M. Table 4 Bud formation of mature groum on the basal medium containing DCMU and sucrose under continuous illumination of white, blue, red and far-red light and in darkness''* Light condition White White Blue Blue Green Green Red Red Far-red Far-red Presence (+) or absence ( ) of 2% sucrose Observed 60 days after the start of culture. * DCMU, -* M cultured with bud(s) (%) 0 (0.0) 16 (80.0) 4 (.0) 35 (92.1) 4 (28.6) 11 (84.6) 1 (5.3) 7 (35.0) 3 (21.5) 13 (92.9) 2 (12.5) 18 (72.0) Average number of buds per O.0±0.OO 1.0±0. 0.2± ± ± ± ± Downloaded from at Penn State University (Paterno Lib) on September 16, 16

6 796 Y. Kato Effect of AT It is known that a permanent reduction in the leaf's ability to manufacture chlorophyll occurs when AT is present in the medium {). AT at 2 X ~ 4 M and in combination with DCMU at ~ 5 M inhibited bud formation almost completely on both mature and young leaf (Table 5). Irreversible chlorosis occurred in leaf treated with AT. The inhibitory effect of AT was slightly reduced by the addition of sucrose to the medium. Effect of absorption of C0 z by KOH In order to absorb carbon dioxide, was put in the central well of the culture flask. The experimental results showed the importance of released CO2 for bud formation in both light and darkness (Table 6). The inhibition in a CO2-free atmosphere was reversible. When, growing on a basal medium containing no sugar, were placed in light, deprived of CO2 for about two months, and subsequently exposed to air, buds were formed in 5 or 6 weeks. When were grown on a medium containing 2% sucrose under a CO2-free atmosphere, adventitious buds were produced much as in the control. These results Table 6 Exp. no. I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI Influence of the presence of KOH as an absorbent of CO 2 and sucrose on bud formation in leaf "^ Central well content Presence (+) or absence (-) of 2% sucrose Source of Light Mature leaevs Number of cultured with buds 16 (80.0) 13 (92.9) 12 (75.0) 4 (22. 2)* 6 (13.9)* 0 (0.0)* 9 (60.0) 5 (83.3) 11 (73.3) (33.3)* 12 (54.5)* 5 (45.5)* 0 (0.0)* 0 (0.0)* 13 (81.3) 17 (85.0) Average number of buds per 1.1 ± ± ± ± ± ± ± ± ± ± ±0.25 * Observed 60 days after the start of culture. * Experiments were conducted under continuous illumination by white light or in darkness. * Statistically different from the control. Downloaded from at Penn State University (Paterno Lib) on September 16, 16

7 Role of photosynthesis on bud formation 797 Table 5 Inhibition of bud formation on young and mature leaf by AT and DCMU and in combination with sucrose'' b Additives to the basal medium None AT (2 X - 4 M) AT (2 x - 4 M) -(-sucrose DCMU (- S M) AT (2X~»M) +DCMU (- 5 M) Source of cultured with buds 28 (93. 3) 52 (94. 5) 0 (0.0) 0 (0.0) 2 (8.3) 1 (4.2) 16 (53.3) 7 (33.3) 0 (0.0) 0 (0.0) " Observed 60 days after the start of culture. * All the experiments were conducted under continuous illumination by white light. Average number of buds per 1.6± ± ± ± ± ± ± ±0.00 Table 7 Influence of citrate on bud formation in mature leaf under continuous illumination by white light and in darkness " Exp. no. co^firi on the basal medium Additives to I II III IV Light Light None Citrate (" 2 M) None Citrate (" 2 M) " Observed 60 days after the start of culture. Number of with bud(s) cultured (%) (0.0) (90.0) 18 (90.0) 9 (81.8) Average number of buds per 0.0± ± ±0.21 Average plant height-mm 0.0± ± ±0.33 suggest that the presence of available released CO2 or sucrose in the medium is an important factor for bud formation. Effect of organic acids The organic acids, citrate, malate and succinate, were supplied at a concentration of ~ 2 M in the basal medium. Citrate could substitute for sucrose in darkness (Table 7). L-Malate could replace sucrose partially in both light and darkness. Succinate caused the death of the leaf. Downloaded from at Penn State University (Paterno Lib) on September 16, 16 Discussion The data reported in the present paper suggested that light acts for bud formation through some photosynthetically dependent process. The experiments using DGMU and sucrose showed that photosynthesis or carbohydrate availability

8 798 Y. Kato plays an important role in the bud formation process. In darkness, bud formation is induced if sugar is added to the nutrient medium (7). Furthermore, addition of sugar to CO2-starved resulted in bud formation. Lack of photosynthesis and shortage of food in the leaf inhibited this. It was found in the previous studies (, 15) with tobacco callus cultures that carbohydrate metabolism plays a dramatic role during organogenesis. A strong correlation between starch accumulation, the rate of respiration in the leaf tissue and bud formation was observed (15). It is reasonable to assume that organ formation in Heloniopsis leaf is closely similar to that in tobacco callus cultures. Carbohydrates in the leaf tissue may be utilized during formation and growth of shoot primordia and their development into leafy shoots. The auxin/cytokinin interaction hypothesis by Skoog and Miller (13) has been tested and proved in various ways on bud formation on excised Begonia and Bryophyllum leaf (1-4). In Heloniopsis leaf, the importance of these growth regulators has also been confirmed and discussed (6). However, these previous studies on leaf have been made under continuous light or lightdark cycles. Therefore, the contribution of photosynthesis to bud formation should be taken into consideration. Further research is necessary for a quantitative analysis which may show a parallelism between budding and photosynthetic activity of the leaves in Btyophyllum and Begonia. The production of buds was considerably influenced by leaf age and environmental conditions before and after excision. Since it is known that changes in the endogenous growth-regulating substance contents of the leaf-blade (11) and in the photosynthetic acitvity (5) are related to the leaf age, the production of buds may be naturally influenced by these factors. I wish to thank Mrs. N. Nakamura and Mrs. N. Ozawa for their technical assistance. References ( 1 ) Fonnesbech, M.: The influence of NAA, BA and temperature on shoot and root development from Begonia X cheimaniha petiole grown in vitro. Physiol. Plant. 32: (1974). ( 2) Heide, O. M.: Effect of 6-benzylaminopurine and 1-naphthaleneacetic acid on the epiphyllous bud formation in Btyophyllum. Planta 67: (1965). ( 3) Heide, O. M.: Photoperiodic effects on the regeneration ability of Begonia leaf cuttings. Physiol. Plant. 18: (1965). (4) Heide, O. M.: The role of cytokinin in regeneration processes. In Hormonal Regulation in Plant Growth and Development. Edited by H. Kaldewey and Y. Vardar. Proc. Adv. Study Izmir, p , (5) Hernandez-Gil, R. and M. Schaedle: Functional and structural changes in senescing Populus dcltoidcs (Partr.) chloroplasts. Plant Physiol. 51: (1973). ( 6 ) Kato, Y.: Bud formation on excised Heloniopsis leaf fragments. Effects of leaf age and the midrib. Plant & Cell Physiol. 15: (1974). ( 7 ) Kato, Y.: Induction of adventitious buds on undetached leaves, excised leaves and leaf fragments of Heloniopsis orientalis. Physiol. Plant. 42: (1978). ( 8) Kato, Y. and S. Kawahara: Bud formation in leaves, leaf fragments and midrib pieces of Heloniopsis orientalis (liliaceae). Planta 7: (1972). ( 9 ) Murashige, T. and F. Skoog: A revised medium for rapid growdi and bioassays with tobacco callus cultures. Physiol. Plant. 15: (1962). Downloaded from at Penn State University (Paterno Lib) on September 16, 16

9 Role of photosynthesis on bud formation 799 () Pyfrom, H. T., D. Appleman and W. G. Heim: Catalase and chlorophyll depression by 3- amino-l,2,4-triazole. Plant Physiol. 32: (1957). (//) Shoji, K., F. T. Addicott and W. A. Swets: Auxin in relation to leaf blade abscission. Plant Physiol. 26: (1951). (12) Sironval, C.: Action of day-length upon the formation of adventitious buds in Bryophyllum tubiflonm Harv. Nature 178: (1956). (13) Skoog, F. and C. O. Miller: Chemical regulation of growth and organ formation in plant tissue cultured in vitro. Symp. Soc. Exp. Biol. 11: (1957). () Thorpe, T. A.: Carbohydrate availability and shoot formation in tobacco callus culture. Physiol. Plant. 30: (1974). (75) Thorpe, T. A. and D. D. Meier: Starch metabolism, respiration and shoot formation in tobacco callus cultures. Physiol. Plant. 27: (1972). Downloaded from at Penn State University (Paterno Lib) on September 16, 16

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