Jan 31, 1978 - plant stage did influence thesubsequent root formation. Cuttings from stock plants ... Light and Light-measuring Equipment. All irradiances were.
Plant Physiol. (1978) 61, 975-979
Influence of the Irradiance on Carbohydrate Content and Rooting of Cuttings of Pine Seedlings (Pinus sylvestris L.)1 Received for publication August 30, 1977 and in revised form January 31, 1978
JURGEN HANSEN,2'3 LARS-HELGE STROMQUIST, AND ANDERS ERICSSON Department of Plant Physiology, University of Umea, S-901 87 Ume'a, Sweden ABSTRACT
rather complex. It has been suggested (9, 10) that the increasing level of irradiance inhibits root initiation in pea cuttings through a supraoptimal carbohyrate content in relation to the endogenous auxin level. In contrast to the decreasing number of roots in pea cuttings from intact stock plants grown at increasing irradiances (9, 10, 27), it was demonstrated that increasing irradiances to decotyledonized stock plants (27) and to Chrysanthemum stock plants (7) stimulated the subsequent formation of roots in cuttings. Although there are big differences in root number the similarity in the pattern of reaction to increasing irradiances in decotyledonizod pea plants (27) and in Chrysanthemum plants (7) indicates common features in the process of root formation. The differential responses to radiant energy in different plants (7, 9, 10) thus may be the result of differences in content and distribution of important metabolic
Seedlings of Pinus sylvestris L. were grown for 6 weeks at an irradiance of either 8 or 40 watts per square meter in a controlled environment room. Cuttings from these plants were rooted in tap water for 75 days at either 8 or 40 watts per square meter. The photoperiod was 17 hours. During the first 30 days of the rooting period quantitative changes in carbohydrates were recorded in cuttings from the different treatments. The carbohydrate contents of the cuttings were mainly regulated by the irradiance during the stock plant stage and generally a higher carbohydrate level was found in cuttings from stock plants grown at 40 watts per square meter. The irradiance during the rooting period had only minor effects on the time course of root formation, whereas the irradiance during the stock plant stage did influence the subsequent root formation. Cuttings from stock plants grown at 8 watts per square meter rooted faster and with higher frequency than those from stock plants grown at 40 watts per square meter. These results are discussed in relation to the mentioned irradiance effects on carbohydrate content.
products. The purposes of this investigation were to study root formation in pine cuttings and the variation in endogenous carbohydrates during rooting as affected by the irradiance to stock plants and
cuttings.
Since the suggestion by Kraus and Kraybill (13) that a relationship exists between carbohydrate content and rooting of cuttings, this hypothesis has been the subject of several investigations (e.g. 4, 17). Nevertheless, the role of carbohydrates in rooting of cuttings is obscure. Stimulatory effects of carbohydrates have been reported (2, 12, 15, 20), as well as inhibitory effects (16, 17). The variation in carbohydrate effects on root formation indicates interactions with other factors controlling root formation. From the reports by Lovell et al. (16) and Howard and Sykes (12) we concluded that the effects of exogenously supplied carbohydrates are controlled by the irradiance and by endogenous carbohydrate sources. An interaction between carbohydrates and auxin in root formation (1, 8, 20) could be a plausible explanation of the diversity in carbohydrate effects reported. In addition, the findings by Hansen and Eriksen (10) and Fischer and Hansen (7) that rooting of cuttings is influenced by irradiance prevalent during the stock plant stage demonstrate the importance of strict environmental control for obtaining reproducible results in rooting experiments. Since radiant energy influences the content (26) and translocation (21) of growth hormones as well as assimilate production, the influence of irradiance on adventitious root formation becomes ' This investigation was supported by a Danish Natural Science Research Council postdoctoral fellowship to J. H. This work was also supported by the J. C. and Seth M. Kempe's Memorial Foundations and by the Swedish Council for Forestry and Agriculture Research. 2 Present address: Institute of Biology and Geology, University of Troms0, P.O. Box 790, N-9001 Troms0, Norway. 3To whom reprint requests should be made.
MATERIALS AND METHODS Growing Conditions for Stock Plants. Seeds of Pinus sylvestris L. (harvested in 1972 in the central part of Sweden, latitude 63°40'N, longitude 17020'E, altitude 210 m) were grown for 6 weeks in a controlled environment room. All of the seeds were germinated at an irradiance of 40 w m-2 (400-715 nm) for about I week. For the remaining 5 weeks half of the seedlings were grown at an irradiance of 8 w m-2 incident at plant level and the other half at 40 w m2. The growing medium consisted of peat (Solmull, Hasselfors Bruks AB, Sweden) and the plants were fertilized twice during the growing period and watered when required. The fertilizer was a 1:1000 solution of Wallco fertilizer (Astra-Wallco AB, Sweden). The photoperiod was 17 hr, the air temperature 20 C, and the relative humidity 80%o. Light and Light-measuring Equipment. All irradiances were determined with a calibrated thermophile (Hilger Watts) and a galvanometer (Kipp and Zonen, type AL 1). The measurements were corrected for radiant energy above 715 nm by means of a Schott-Jena RG 715 filter. The reduced level of irradiance of 8 w m-2 was accomplished with white cheesecloth. Measurements of the spectral distribution (Fig. 1) were made with a quanta spectrometer (QSM-2500, Techtum Instrument, Umea, Sweden). The radiant source consisted of Osram HQI 400 w-70 tubes. Treatment of Cuttings and Growing Conditions. Six weeks after sowing the seedlings were excised just above the soil surface and selected for uniformity. The length of the hypocotyl was approximately 2 cm. The cuttings were transplanted to 1-cm-thick Styrofoam plates floating on aerated tap water. Cuttings from stock plants grown at 40w m2 were rooted at either 8 or 40 w m-2 and cuttings from stock plants grown at 8 w m-2 were consequently rooted at either 8 or 40 w m-2. Temperature, photoperiod, and humidity were the same as for the stock plants.
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Plant Physiol. Vol. 61, 1978
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700 600 WaveWngth, nm FIG. 1. Relative spectral energy distribution of the radiant energy used for growing stock glants and for rooting of cuttings. Distribution of radiant energy at 40 w m (-) and after reduction of the irradiance to 8 w m-2 with white cheesecloth (- -- -). For comparison the signal at A = 546 nm -). The radiation for 8 w m-2 was amplified to the level of 100o --source consisted of Osram HQI 400 w-70 tubes. Within the wavelength range of 400 to 715 nm the plants received 17.4 x 10' quanta cm 2sec-1 at the irradiance level of 40 w m-2 and 3.7 x lO'5 quanta cm-2sec-' at the level of 8 w m-2, as measured with the QSM-2500 quanta spectrometer. 400
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FIG. 2. Appearance of cuttings at the day of excision from 6-week-old seedlings of P. sylvestris L. Left: stock plants grown at 40 w m 2; right: stock plants grown at 8 w m Table I.
Effect of Irradiance on Fresh and Dry Weights of Cuttings of Pinus
sylvestr8s
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Cuttings were excised and rooeda Seedlings were grown for 6 weeks at 8 or 40 wm described in Materials and Methods. Time is measured from the beginning of the rooting period. The data are given in mg per cutting representing the mean of 40 cuttings.
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59.8 20.9 129.0 38.8 18.6 65.7 61.6 At intervals of 2 to 4 days during a rooting period of 75 days 56.8 23.1 50.6 65.2 155.0 65.0 22.5 63.3 60.3 67.6 27.8 151.0 23.0 73.9 the percentage of cuttings that rooted and the number of roots/cutting were recorded. Because of the special floating system these measurements could be done without any injury to the RESULTS cuttings. The number of cuttings/irradiance combination was 120 Influence of Irradiance on Growth of Stock Plants. At the time and the whole experiment was repeated with approximately the same number of cuttings. Statistical evaluation of the rooting cuittings were excised differences in plant size due to the different irradiances could be observed. The plants grown at 40 w m-2 had results was based on calculation of 95% confidence limits. Treatment of Plant Material for Carbohydrate Determination. approximately twice as many needles (Fig. 2) and had more rigid At different times during the rooting period cuttings from a hypocotyls than those grown at 8 w m-2. The differences are also parallel experiment were randomly collected for carbohydrate indicated by their fresh and dry wt (Table I). Influence of Irradiance on Carbobydrate Content. During rootdeterminations. Two replicates of 20 cuttings each were collected from each irradiance combination at the time of excision and after ing the carbohydrate content increased in all cuttings regardless 5, 10, 15 and 30 days. After determination of the fresh wt the plant of the irradiance treatments. Higher contents of all carbohydrates material was immediately dried at 60 C for at least 72 hr and then measured were found in cuttings from plants grown at 40 w m-2 ground in a ball mill (Retsch Muhle) for 10 min. The ground than in cuttings from plants grown at 8 w m2 (Fig. 3, A-F). The plant material was kept in a desiccator over silica gel until analysis. differences in carbohydrate content caused by the different irraDetermination of Soluble Carbohydrates and Starch. Two par- diance treatments of the stock plants were large and persistent allel percolations from each replicate sample were made to check during the first 30 days of the rooting period. The level of the reproducibility of the percolation procedure. The procedure irradiance during the rooting period had substantially less influwas as described by Hansen and M0ller (1 1). The amount of plant ence on the carbohydrate content of the cuttings. Consequently, material/analysis was 40 to 50 mg dry wt and the volume of 80% cuttings taken from stock plants grown at 8 w m-2 and rooted at (v/v) aqueous ethanol for percolating the soluble carbohydrates either 8 w m2 [8 w m-2, 8 w m-1 or 40 w m-2 [8 w m-2 40 w m-2 was 22 ml. The ethanol percolates were made up to 25 ml. After contained similarly low carbohydrate levels. The [40 w m2, 40 w removal of soluble carbohydrates the residue containing the starch m-2] cuttings had the highest carbohydrate content, and the [40 w fraction was percolated with 30 ml of a 35% (v/v) aqueous m2, 8 w m-2 cuttings were with a few exceptions intermediate in this respect. perchloric acid solution. Influence of Iffadiance on Root Formation. Different levels of Carbohydrates present in the ethanol percolate were determined quantitatively by GLC. Two ml of the percolate with and without irradiance, especially during the period of stock plant growth, had addition of 100 ,u of aqueous myo-inositol solution (2 mg/ml) as pronounced effects on the rooting of cuttings (Fig. 4, A and B). internal standard were freeze-dried. The gas chromatograph was The first visible roots appeared after 22 days in the [8 w m2, 8 w a Pye Unicam 104 equipped with a hydrogen flame ionization m-21 material, whereas in the [40 w m-2, 40 w m-21 material the first root penetration was observed after 31 days. detector. The procedure was as described by Ericsson et al. (5). The differences in rooting were significant and persistent during Each perchloric acid percolate was analyzed two times quantithe rooting period (Fig. 4, A and B). The highest number of tatively for starch as described by Hansen and M0ller (I 1).
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ROOT FORMATION: LIGHT AND CARBOHYDRATES
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15 15 20 25 30 0 5 10 20 25 30 Time, days Time, days Time, days FIG. 3. Influence of irradiance on the content of soluble carbohzdrates and starch in cuttings of P. sylvestris L. Stock plants were grown at 8 w m-2 (O and 0) or 40 w m-2 (E and U). Cuttings were rooted at 8 w m- (O and l) or 40 w m-2 (0 and U). Each point is the mean of four determinations, two determinations from each of two replicate percolations. Curves represent the mean of measurements on two replicate plant samples. Time is measured from the beginning of the rooting period.
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roots/cutting and the highest percentage of cuttings with roots were found in cuttings rooted at 8 w m-2 and which originated from stock plants grown at 8 w m2. Cuttings from stock plants grown at 40 w m-2 and rooted at 40 w m-2 had the lowest number of roots as well as the lowest rooting percentage. The treatments [8 w m2, 40 w m-2] and [40 w m-2, 8 w m-2 were intermediate in their effects on rooting with the [8 w m-2, 40 w m-2] being superior to [40 wm,2 8 w m-. The results demonstrate that the rooting process was more influenced by the level of irradiance during the stock plant stage than by the irradiance during the period of root formation. DISCUSSION A reduction in the number of roots with an increase in the irradiance during stock plant stage is in accordance with results obtained with Pisum sativum L. cv. Alaska (9, 10, 14) and Dahlia variabilis (3). An opposite effect on root formation of increasing the irradiance, however, was found with Chrysanthemum morifol-
ium Ramat (7). Due to the great many physiological processes influenced by radiant energy several explanations are possible to the obtained results. Hansen and Eriksen (10) suggested that the irradiance effect in Pisum was mediated through carbohydrates and/or hormones. It was proposed that a carbohydrate level exceeding a certain limit could result in a reduction of root formation. It is probable that the higher and steadily increasing carbohydrate content in cuttings from stock plants grown at the high irradiance is supraoptimal for root formation. The increase in carbohydrate content in the cuttings during the rooting period is consistent with results obtained with Cucurbita pepo L. cotyledon cuttings (18). The increase in carbohydrate content in the Pinus cuttings during the rooting period thus may be due to the absence of a sink, i.e. the roots. A carbohydrate build-up can be expected although photosynthesis is rapidly declining in all cuttings regardless of the irradiance pretreatments (Brunes, unpublished results). The role of carbohydrates in rooting of cuttings has most intensively been studied by Loveli and co-workers (16, 17) in
HANSEN, STROMQUIST, AND ERICSSON
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FIG. 4. Influence of irradiance on root formation in P. sylvestris L. Stock plants were grown at 8 w m-2 (O and *) or 40 w m-2 (0 and U). Cuttings rooted at 8 w m-2 (O and 0) or 40 w m-2 (0 and 3). Mean number of roots and the percentage of rooting based on the total number of cuttings were calculated at intervals of 2 to 4 days. Each treatment covered about 250 cuttings. Results are the mean of two replicate experiments. Vertical bars denote 95% confidence limits.
cotyledon cuttings of Sinapis alba L. and Raphanus sativus L. Exogenously supplied carbohydrates reduced root formation and rapid increases in endogenous carbohydrate levels were observed (17). Sucrose was most inhibitory followed by glucose and fructose. The delay in appearance of the first root as well as the lower rooting percentage of the Pinus cuttings with the higher contents of different carbohydrates are, therefore, in accordance with the results by Lovell et al. (17). Surprisingly, different irradiances during the rooting period had no significant effect on either the number of roots/cutting or the percentage of cuttings with roots. These results are not immediately in agreement with the observations (12, 19) that rooting is increased with increasing irradiance during root formation. However, in these experiments rooting was improved by sucrose supplied to cuttings grown under low irradiance (12) or to dark-grown or DCMU-treated, light-grown cuttings (16). The stimulation of increasing irradiances in the absence of sucrose (12, 19), therefore, indicates a suboptimal supply of metabolic products for root formation in these plants. The present results with Pinus support the theory that the physiological status of the stock plant at the time that cuttings are excised is of utmost importance for the subsequent rooting process (9, 10, 27). The relatively small influence on carbohydrate content of different irradiances during the rooting period compared to the large effect of different irradiances given to stock plants demonstrates the importance of controlled growing conditions for stock plants designated for rooting experiments. Although it is generally accepted that a carbohydrate source is essential for root formation, the significance of different carbohydrates in this process is obscure. The basic knowledge of the importance of different carbohydrate constituents of a plant is restricted primarily because of the lack of specific and convenient analytical methods. More information on the distribution of the individual carbohydrates and their rates of turnover is necessary in order to evaluate the importance of individual carbohydrates in root formation. Sucrose is considered an essential carbohydrate for the development of roots in stem cuttings (1, 12, 27) as in plant parts grown in vitro (8, 15). Among a wide range of different carbohydrates tested (15), sucrose was found to be most efficient in stimulating root formation in dark-grown pea stem segments. In the Sinapis and Raphanus rooting system a number of different carbohydrates
all supplied with the same molarity to light-grown cotyledons revealed differential but inhibitory effects (17). However, in darkgrown cotyledons and in cotyledons treated with the photosynthetic inhibitor DCMU, sucrose stimulated root formation (16). The level of sucrose in the petioles of cotyledon cuttings of S. alba was found to be relatively low even in cuttings incubated in sucrose solutions (17). In agreement with this finding, sucrose was found to be present at a very low level among the carbohydrates found in the pine cuttings. Glucose has also been reported to stimulate the rooting process (15, 20). In comparison with sucrose (15), glucose and fructose were reported less active in stimulating root formation. The efficiency of glucose, however, was dependent on whether glucose was supplied to the in vitro grown stock plants or only after cuttings were excised. These experiments were performed in darkness (15). The reported interaction between carbohydrates and hormones in adventitious root formation (1, 8, 20) makes a discussion based solely on carbohydrates inadequate. Radiant energy has been reported to influence auxin metabolism (26) and auxin translocation (21) and it is therefore possible that the irradiance effect on rooting is mediated partly through auxin, as this hormone is an important factor in adventitious root formation (6). An auxincarbohydrate interaction (1, 8, 20) offers a possible explanation of different rooting responses to light in different plant species (7, 10). Assimilate production, auxin-producing capacity, and auxin translocation are influenced by the irradiance and might determine the carbohydrate-auxin balance and with it the rooting potential of the cutting. The present results are, therefore, not necessarily inconsistent with the findings that good root formation is related to a high endogenous carbohydrate content (12, 13, 24). It is also possible that the differences in rooting of cuttings from differently pretreated stock plants could be related to irradianceinduced differences in the developmental stage. It has been shown that the light conditions during plant growth could affect the gradual changes from the juvenile to the adult phase (23). Cuttings from young plants are generally rooted easier than cuttings from adult plants (22, 25). The irradiance-induced differences in root formation indicate that the process of adventitious root formation is a complex system of events which interacts with the environment not only during the rooting process itself, but also during the stock plant stage.
Plant Physiol. Vol. 61, 1978
ROOT FORMATION: LIGHT AND CARBOHYDRATES
Our results clearly demonstrate that the environmental conditions during stock plant stage are of greater importance for the subsequent rooting in cuttings than is commonly acknowledged. Acknowledgments-The authors wish to thank L. Eliasson and A. Dunberg for critically reading the manuscript. LITERATURE CITED 1. ALTMAN A, PF WAREING 1975 The effect of IAA on sugar accumulation and basipetal transport of "C-labelled assimilates in relation to root formation in Phaseolus vulgaris cuttings. Physiol Plant 33: 32-38 2. BACHELARD EP, BB STOWE 1962 A possible link between root initiation and anthocyanin formation. Nature 194: 209-210 3. BIRAN 1, AH HALEVY 1973 Stock plant shading and rooting of Dahlia cuttings. Scientia Horticulturae 1: 125-131 4. BOUILLENNE R, F WENT 1933 Recherches experimentales sur la neoformation des racines dans les plantuies et les boutures des plantes superieures. Ann Jard Bot Buitenzorg 43: 25-202 5. ERicssoN A, J HANSEN, L DALGAARD 1978 A routine method for quantitative determination of soluble carbohydrates in small samples of plant material with gas liquid chromatography. Anal Biochem. In press 6. ERIKSEN EN, S MOHAMMED 1974 Root formation in pea cuttings. II. The influence of indole3-acetic acid at different developmental stages. Physiol Plant 30: 158-162 7. FISCHER P, J HANSEN 1977 Rooting of Chrysanthemum cuttings. Influence of irradiance during stock plant growth and of decapitation and disbudding of cuttings. Scientia Horticulturae 7: 171-178 8. GREENWOOD MS, GP BERLYN 1973 Sucrose-indole-3-acetic acid interactions on root generation by Pinus lambertiana embryo cuttings. Am J Bot 60: 42-47 9. HANSEN J 1976 Adventitious root formation induced by gibberellic acid and regulated by the irradiance to the stock plants. Physiol Plant 36: 77-81 10. HANSEN J, EN ERIKSEN 1974 Root formation of pea cuttings in relation to the irradiance of the stock plants. Physiol Plant 32: 170-173 11. HANSEN J, I M0LLER 1975 Percolation of starch and soluble carbohydrates from plant tissue for quantitative determination with anthrone. Anal Biochem 68: 87-94
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12. HOWARD BH, JT SYKES 1966 Regeneration of the hop plant (Humulus lupulus L.) from softwood cuttings. II. Modification of the carbohydrate resources within the cutting. J. Hort Sci 41: 155-163 13. KRAUS EJ, HR KRAYBILL 1918 Vegetation and reproduction with special reference to the tomato. Oreg Agric Exp Sta Bull 149 14. LEROUX R 1965 Etude de la rhizogenese de segments d'epicotyles de pois (Pisum sativum L.) en fonction de la lumiere re,ue par les plantules sur lesquelles ils ont etc preleves. CR Hebd Seances Acad Sci Ser D Sci Nat 261: 5609-5611 15. LEROUX R 1973 Contribution a l'etude de la rhizogenese de fragments de tiges de pois (Pisum sativum L.) cultives in vitro. Rev Cytol Biol Veg 36: 1-132 16. LOVELL PH, A ILLSLEY, KG MOORE 1972 The effects of light intensity and sucrose on root formation, photosynthetic ability, and senescence in detached cotyledons of Sinapis alba L. and Raphanus sativus L. Ann Bot 36: 123-134 17. LOVELL PH, A ILLSLEY, KG MOORE 1974 Endogenous sugar levels and their effects on root formation and petiole yellowing of detached mustard cotyledons. Physiol Plant 31: 231-236 18. MOHAMMAD AMS, Y AL-MASHHADANI 1976 The effect of root formation on the levels of protein, chlorophyll, RNA, DNA and carbohydrates in excised cotyledons of Cucurbitapepo. Physiol Plant 37: 195-199 19. MOORE K, P LOVELL 1970 Control of rooting and the pattern of senescence in detached white mustard cotyledons. Physiol Plant 23:985-992 20. NANDA KK, MK JAIN, S MALHOTRA 1971 Effect of glucose and auxins in rooting etiolated stem segments of Populus nigra. Physiol Plant 24:387-391 21. NAQVI SM, SA GORDON 1967 Auxin transport in Zea mays coleoptiles. 11. Influence of light on the transport of indoleacetic acid-2-'4C. Plant Physiol 42: 138-143 22. PORLINGIS IC, I THERIOS 1976 Rooting response of juvenile and adult leafy olive cuttings to various factors. J Hort Sci 51: 31-39 23. SCHAFFALITZKY. M DE MUCKADELL 1959 Investigations on aging of apical meristems in woody plants and its importance in silviculture. Forstl Fors0gsves Dan 25: 307-455 24. STOLTZ LP 1968 Factors influencing root initiation in an easy- and a difficult-to-root Chrysanthemum. Proc Am Soc Hort Sci 92: 622-626 25. THIMANN KV, AL DELISLE 1939 The vegetative propagation of difficult plants. J Arnold Arbor Harv Univ 20: 116-136 26. TILLBERG E 1974 Levels of indol-3yl-acetic acid and acid inhibitors in green and etiolated bean seedlings (Phaseolus vulgaris). Physiol Plant 31: 106-111 27. VEIERSKOV B, J HANSEN, AS ANDERSEN 1976 Influence of cotyledon excision and sucrose on root formation in pea cuttings. Physiol Plant 36: 105-109
