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The Levels of Endogenous Cytokinins in Daffodil Bulbs Stored under Different Environmental Conditions
Department of Botany, University of Natal, Pietermaritzburg 3200, South Africa
The Levels of Endogenous Cytokinins in Daffodil Bulbs Stored under Different Environmental Conditions J. VAN STADEN With 5 figures Received September 3, 1977 . Accepted September 21, 1977
Summary The endogenous cytokinin levels in bulbs of daffodil (Narcissus pseudonarcissus L.) are affected by moisture and temperature. Keeping bulbs in cold moist conditions forces them to grow and also results in a large increase in their cytokinin levels. Most of the cytokinins are present in the roots which develop on the bulbs. It is suggested that the production of these compounds in the roots under cold moist conditions and their subsequent transport to the shoots has a marked effect on bulb growth. Key words: Cytokinins, bulbs, daffodil, Narcissus pseudonarcissus.
Introduction REES (1972) expressed the opinion that the growth originating from a bulb or corm is equivalent to that derived from a large seed. In both cases the presence of ample food reserves allows the plant to survive unfavourable environmental conditions. This survival, as occurs in buds and seeds, is made more efficient by the fact that bulbs and corms may pass through a period of true physiological dormancy. As is the case in seed dormancy (WAREING and SAUNDERS, 1971) various types of bulb dormancy can be distinguished (RUDNICKI, 1974). These types of bulb dormancy may or may not be hormonally controlled. Often these dormant bulbs or corms can be induced to resume normal growth by maintaining them at low temperatures for certain periods of time (GINZBURG, 1974; RUDNICKI, 1974). Although many attempts have been made to replace the low temperature requirement, particularly by applying gibberellins which occur naturally in bulbs (AUNG et aI., 1969; RUDNICKI, 1974), none of these treatments has produced truly satisfactory results (HALEVY and SHaUB, 1964; AUNG and DE HERTOGH, 1967). In many respects this situation is analogous to that in seeds which require a period of stratification for the breaking of dormancy (WEBB ('t a!., 1973; RUDNICKI et aI., 1973). Different hormones may be active at different stages in the overall process of dormancy release in seeds (WEBB et aI., 1973; BORKOWSKA and RUDNICKI, 1975; BROWN and VAN STADEN, 1975). In the
z. P/lanzenphysiol. Bd. 86. S. 323-330. 1978.
324
].
VAN STADEN
case 'Of hyacinth bulbs the situation appears to be similar, as the endogenous, auxin gibberellin, cytokinin, and abscisic acid levels were closely correlated with the stage of development reached, and with the conditions under which the bulbs were stored (RUDNICKI and NOWAK, 1976). It is essential to obtain more information about the role of endogenous hormones in bulbs. Such information would enable growers to fully exploit the use of hormones to induce growth if these compounds could be used to substitute for certain environmental requirements necessary for the resumption of growth. In view of the knowledge concerning the involvement of cytokinins in seed dormancy (WAREING and SAUNDERS, 1971) and the recent report that applied cytokinins enhance the growth of dormant gladiolus corms (GINZBURG, 1974), it was decided to investigate whether or not these compounds are present in daffodil bulbs and to what extent the endogenous levels of the cytokinins change under different storage conditions. Material and Methods Plant material Samples of uniform daffodil bulbs (Narcissus pseudonarcissus L.) cv. Unsurpassable, were stored dry and moist in vermiculite in the dark at 26°C and 10 °C respectively. Ten bulbs, weighing 500 g at the commencement of the experiment, were used for each treatment. Bulbs were analysed for cytokinins at zero days and after 28 and 56 days of storage. In one experiment the whole bulb, irrespective of whether or not root and/or shoot growth occurred, were analysed. In a second experiment the roots plus the basal plates were analysed separately from the bulb scales. Extraction and bioassay for cytokinins The ten bulbs of each treatment (initial weight 500 g) were homogenized in 500 ml 80 Ofo ethanol and extracted at room temperature for 48 h. After filtration the residue was washed with a further 500 ml 80 % ethanol and the ethanolic extracts combined. These combined extracts were taken to dryness in vacuo at 35°C and the residue redissolved in 100 ml 80 0/0 ethanol. The equivalent of 50 g fresh bulb material was subsequently used for the extraction and purification of cytokinins. The pH of the ethanolic extracts were adjusted to 2.5 with HCI whereafter they were filtered through Whatman No. 42 filter paper. The filtrate was then passed through a Dowex 50W-X8 cation exchange resin (H+ form; 200-400 mesh; column 2X 10 cm) at a flow rate of 10 mllh. The column was washed with 50 ml 80 0/0 ethanol. Compounds responsible for cell division activity were eluted from the column with 250 ml 5 N NH 40H. The ammonia was removed under vacuum and the residue redissolved in 4 ml 80 Ofo ethanol. These Dowex purified extracts were loaded onto Whatman No. 1 chromatography paper and separated with iso-propanol: 25 Ofo ammonium hydroxide: water (10 : 1 : 1 v/v). The chromatograms were dried in a stream of air, divided into ten equal R f strips and these then assayed for cytokinin activity with the soybean callus bioassay. Intact bulbs and bulb scales were assayed on 30' ml of medium in 50 ml erlenmeyer flasks while root material was assayed on 15 ml medium in 25 ml culture flasks.
Results and Discussion Temperature had a pronounced effect on the growth and development of daffodil bulbs. Most rapid growth occurred at 10°C, where after 28 days, roots were well Z. Pflanzenphysiol. Bd. 86. S. 323-330. 1978.
Cytokinins in daffodil bulbs
325
developed irrespective of whether or not the bulbs were stored dry or moist. At 26 °C root development was slow (Fig. 1). After 56 days of storage the root systems and the shoots of bulbs stored at low temperature (10 °C) in moist vermiculite were well developed (Fig. 2). Where the bulbs were stored dry (10 °C) root development had not advanced further than was the case after 28 days of storage. Visible signs of shoot growth could however be observed. Bulbs stored moist at 26 °C for 56 days had well developed root systems and shoot growth was taking place. Those bulbs stored dry at the higher temperature (26 °C) did however, not show any visible signs of root development.
Fig. 1: The development of daffodil bulbs stored moist and dry at 10 °C and 26 °C for 28 days. A = 10 °C moist; B = 10 °C dry; C = 26 °C moist; D = 26 °C dry.
Fig. 2: The development of daffodil bulbs stored moist and dry at 10 °C and 26 °C for 56 days. A = 10 °C moi st ; B = 10 °C dry; C = 26 °C moist; D = 26 °C dry. Z. PJlanzenphysiol. Ed. 86. S. 323-330. 1978.
326
]. VAN
STADEN
Low levels of endogenous cytokinins were detected in the intact bulbs at the commencement of the experiment (Fig. 3). Two peaks of cytokinin activity were recorded after paper chromatography. The first was slow-moving and co-chromatographed with zeatin glucoside (Rf 0.2-0.5), while the second was fast-moving and co-chromatographed with zeatin and zeatin riboside (Rf 0.5-0.8). Both quantitative and qualitative changes in the endogenous cytokinins in daffodil bulbs were recorded during the course of storage. The most pronounced increase in cytokinin activity occurred in those bulbs stored at 10°C, After 56 days of storage the activity was highest in those bulbs stored moist (Fig. 4). The recorded increase in activity at low temperature was due to increases in both the slow- and fast-moving cytokinins (Fig. 3). In the bulbs stored in the cold under moist conditions the free base cytokinins, zeatin and zeatin riboside, were responsible for most of the recorded activity. Under dry conditions however, most activity was attributable to compounds that co-chromatographed with the cytokinin glucosides. These derivatives of zeatin and zeatin riboside are considered to be storage forms (PARKER and LETHAM, 1973) and apparently accumulate in plant tissues that are not rapidly growing (VAN STADEN and PAPAPHILIPPOU, 1977). Storage at 26°C had a less pronounced effect on the endogenous cytokinin levels in the bulbs. In the bulbs stored dry the total cytokinin content did not significantly change from that of the 26 C
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Fig. 3: The effect of moisture and temperature on the endogenous cytokinin levels of intact daffodil bulbs stored at 10°C and 26 °C for 28 and 56 days respectively. The equivalent of 50 g plant material was purified on Dowex 50 and the extracts chromatographed with iso-propanol: 25 % ammonium hydroxide: water (10 : 1 : 1 v/v). ZG = zeatin glucoside; ZR = zeatin riboside; Z = zeatin. Z. Pjlanzenphysiol. Bd. 86. S. 323-330. 1978.
Cytokinins in daffodil bulbs 10
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Fig. 4: Total callus yield of intact daffodil bulbs stored both moist and dry for 28 and 56 days at 10 DC and 26°C respectively. Values were obtained by adding the callus yield recorded after paper chromatography. A = 26 DC dry; B = 26 DC moist; C = 10 DC dry; D = 10 DC moist.
control bulbs. Under moist conditions there was, however, an increase in activity after 56 days of storage (Fig 4). This increase was mainly attributable to an increase in those compounds that co-chromatographed with zeatin and zeatin riboside (Fig. 3). From the above results it is noticeable that there appears to be a close correlation between the cytokinin content in the intact plants and the extent to which root development had occurred. As the roots are considered to be major sites of cytokinin synthesis (KENDE, 1964) it was decided to analyse roots and shoots separately in order to establish more closely the distribution of cytokinins within the bulbs. In bulbs stored dry and moist at 10°C most of the cytokinin activity was present in the roots that developed on the bulbs (Fig. 5). In the dry bulbs most of the activity co-chromatographed with zeatin glucoside while in the moist bulbs the major peak of activity chromatographed with zeatin and zeatin riboside. The presence of the storage cytokinins in the roots of dry stored bulbs is probably related to the fact that the endogenous cytokinins synthesized in the roots are not transported to the shoots. As a result of this they are inactivated in the roots where they accumulate. From the Z. Pflanzenphysiol. Bd. 86. S. 323-330. 1978.
328
J. VAN STADEN
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Fig. 5: The endogenous cytokinin levels in the roots and base plates and shoots of daffodil bulbs stored under moist and dry conditions for 56 days at 10 DC. Extracts were purified on Dowex 50 and the extracts chromatographed with iso-propanol: 25 % ammonium hydroxide: water (10 : 1 : 1 v/v). ZG = zeatin glucoside; ZR = zeatin riboside; Z = zeatin.
results it would appear that shoot development is largely dependent on the extent to which roots develop under the different storage conditions and on the synthesis and translocation of cytokinins from these organs. Gibberellins have been shown to increase in bulbs as a result of cold storage and it was suggested that roots may either be sites of synthesis or may provide the precursors for gibberellin production (REES, 1972). AUNG, DE HERTOGH and STABY (1971) have shown that endogenous gibberellin levels in bulbs are affected by moisture and temperature. The highest gibberellin content was recorded in bulbs kept moist at 9 DC. The removal of the base plates of the bulbs considerably reduced the gibberellin levels. From the present data it can be seen that the endogenous cytokinins were produced in the roots and base plates and were affected by moisture and temperature. In seeds, low temperatures have also been shown to increase endogenous cytokinin levels (VAN STADEN, WEBB and WAREING, 1972; BROWN and VAN STADEN, Z. Pjlanzenphysiol. Bd. 86. S. 323-330. 1978.
Cytokinins in daffodil bulbs
1973; DIMALLA and known at present.
VAN STADEN,
329
1977). How this increase is brought about is not
Acknowledgements The financial assistance of the Council for Scientific and Industrial Research, Pretoria, is gratefully acknowledged.
References AUNG, L. H. and A. A. DE HERTOGH: The effect of cold storage and treatment with gibberellic acid on flowering and bulb yield of Dutch iris. Plant & Cell Physio!. 8, 201-205 (1967). AUNG, L. H., A. A. DE HERTOGH, and G. STABY: Temperature regulation of endogenous gibberellin activity and development of Tulipa gesneriana L. Plant Physio!. 44, 403-406 (1969). - - - The alteration of bulb hormones by environmental stimuli. In: Proc. I Int. Symp. on Flowerbulbs. Centre for Agricultural Publishing & Documentation, Wageningen. pp. 156-161 (1971). BORKOWSKA, B. and R. M. RUDNICKI: Changes in the levels of cytokinins in apple seeds during stratification. Fruit Science Reports 2, No.2, 1-16 (1975). BROWN, N. A. C. and]. VAN STADEN: The effect of stratification on the endogenous cytokinin levels of seeds of Pro tea compacta and Leucadendron daphnoides. Physio!. Plant. 28, 388-392 (1973). - - The effect of temperature and various gases on the germination and endogenous hormone levels of seed of Leucadendron daphnoides. Z. Pflanzenphysio!. 75, 31-37 (1975). DIMALLA, G. G. and]. VAN STADEN: The effect of temperature on the germination and endogenous cytokinin and gibberellin levels of pecan nuts. Z. Pflanzenphysio!. 82, 274-280 (1977). GINZBURG, c.: Studies on the role of ethylene in Gladiolus cormel germination. Plant Science Letters 2, 133-138 (1974). HALEVY, A. H. and]. SHOUB: The occurrence of gibberellin-like substances in tulip tulbs (Tulipa sp.). ]. Hort. Sci. 39, 120-129 (1964). KENDE, H.: Preservation of chlorophyll in leaf sections by substances obtained from root exudate. Science N.Y. 145, 1066-1067 (1964). PARKER, C. W. and D. S. LETHAM: Regulators of cell division in plant tissues. XVI. Metabolism of zeatin by radish cotyledons and hypocotyls. Planta 114, 199-218 (1973). REEs, A. R.: The Growth of Bulbs. Academic Press, London (1972). RUDNICKI, R. M.: Hormonal control of dormancy in flower bulbs. 19th Int. Horti. Congress, Warszawa, pp. 187-195 (1974). RUDNICKI, R. M. and]. NOWAK: Studies on the physiology of hyacinth bulbs (Hyacinthus orientalis L.). VI. Hormonal activities in hyacinth bulbs during flower formation and dormancy release.]. expo Bot. 27, 303-313 (1976). RUDNICKI, R. M., M. SANIEWSKI, and D. F. MILLIKAN: The effect of exogenous plant hormones upon the stratification of apple seeds. Proc. Res. Inst. Porn. Ser. E. No.3, 539-552 (1973). VAN STADEN,]. and A. R. PAPAPHILIPPOU: Biological activity of O-tJ-D-glucopyranosylzeatin. Plant Physio!. 60,649-650 (1977). VAN STADEN, ]., D. P. WEBB, and P. F. WAREING: The effect of stratification on endogenous cytokinin levels in seeds of Acer saccharum. Plant a 104, 110-114 (1972).
z. Pjlanzenphysiol. Bd. 86. S. 323-330. 1978.
330
]. VAN STADEN
WAREING, P. F. and P. F. SAUNDERS: Hormones and dormancy. Annu. Rev. Plant Physiol. 22,261-288 (1971). WEBB, D. P., ]. VAN STADEN, and P. F. WAREING: Seed dormancy in Acer: Changes III endogenous germination inhibitors, cytokinins and gibberellins during the breaking of dormancy in Acer pseudoplatanus L. ]. expo Bot. 24, 741-750 (1973). Prof. ]. VAN STADEN, Department of Botany, University of Natal, P.O.B. 375, Pietermaritzburg 3200, Natal, South Africa.
Z. P/lanzenphysiol. Ed. 86. S. 323-330. 1978.
The Levels of Endogenous Cytokinins in Daffodil Bulbs Stored under Different Environmental Conditions J. VAN STADEN With 5 figures Received September 3, 1977 . Accepted September 21, 1977
Summary The endogenous cytokinin levels in bulbs of daffodil (Narcissus pseudonarcissus L.) are affected by moisture and temperature. Keeping bulbs in cold moist conditions forces them to grow and also results in a large increase in their cytokinin levels. Most of the cytokinins are present in the roots which develop on the bulbs. It is suggested that the production of these compounds in the roots under cold moist conditions and their subsequent transport to the shoots has a marked effect on bulb growth. Key words: Cytokinins, bulbs, daffodil, Narcissus pseudonarcissus.
Introduction REES (1972) expressed the opinion that the growth originating from a bulb or corm is equivalent to that derived from a large seed. In both cases the presence of ample food reserves allows the plant to survive unfavourable environmental conditions. This survival, as occurs in buds and seeds, is made more efficient by the fact that bulbs and corms may pass through a period of true physiological dormancy. As is the case in seed dormancy (WAREING and SAUNDERS, 1971) various types of bulb dormancy can be distinguished (RUDNICKI, 1974). These types of bulb dormancy may or may not be hormonally controlled. Often these dormant bulbs or corms can be induced to resume normal growth by maintaining them at low temperatures for certain periods of time (GINZBURG, 1974; RUDNICKI, 1974). Although many attempts have been made to replace the low temperature requirement, particularly by applying gibberellins which occur naturally in bulbs (AUNG et aI., 1969; RUDNICKI, 1974), none of these treatments has produced truly satisfactory results (HALEVY and SHaUB, 1964; AUNG and DE HERTOGH, 1967). In many respects this situation is analogous to that in seeds which require a period of stratification for the breaking of dormancy (WEBB ('t a!., 1973; RUDNICKI et aI., 1973). Different hormones may be active at different stages in the overall process of dormancy release in seeds (WEBB et aI., 1973; BORKOWSKA and RUDNICKI, 1975; BROWN and VAN STADEN, 1975). In the
z. P/lanzenphysiol. Bd. 86. S. 323-330. 1978.
324
].
VAN STADEN
case 'Of hyacinth bulbs the situation appears to be similar, as the endogenous, auxin gibberellin, cytokinin, and abscisic acid levels were closely correlated with the stage of development reached, and with the conditions under which the bulbs were stored (RUDNICKI and NOWAK, 1976). It is essential to obtain more information about the role of endogenous hormones in bulbs. Such information would enable growers to fully exploit the use of hormones to induce growth if these compounds could be used to substitute for certain environmental requirements necessary for the resumption of growth. In view of the knowledge concerning the involvement of cytokinins in seed dormancy (WAREING and SAUNDERS, 1971) and the recent report that applied cytokinins enhance the growth of dormant gladiolus corms (GINZBURG, 1974), it was decided to investigate whether or not these compounds are present in daffodil bulbs and to what extent the endogenous levels of the cytokinins change under different storage conditions. Material and Methods Plant material Samples of uniform daffodil bulbs (Narcissus pseudonarcissus L.) cv. Unsurpassable, were stored dry and moist in vermiculite in the dark at 26°C and 10 °C respectively. Ten bulbs, weighing 500 g at the commencement of the experiment, were used for each treatment. Bulbs were analysed for cytokinins at zero days and after 28 and 56 days of storage. In one experiment the whole bulb, irrespective of whether or not root and/or shoot growth occurred, were analysed. In a second experiment the roots plus the basal plates were analysed separately from the bulb scales. Extraction and bioassay for cytokinins The ten bulbs of each treatment (initial weight 500 g) were homogenized in 500 ml 80 Ofo ethanol and extracted at room temperature for 48 h. After filtration the residue was washed with a further 500 ml 80 % ethanol and the ethanolic extracts combined. These combined extracts were taken to dryness in vacuo at 35°C and the residue redissolved in 100 ml 80 0/0 ethanol. The equivalent of 50 g fresh bulb material was subsequently used for the extraction and purification of cytokinins. The pH of the ethanolic extracts were adjusted to 2.5 with HCI whereafter they were filtered through Whatman No. 42 filter paper. The filtrate was then passed through a Dowex 50W-X8 cation exchange resin (H+ form; 200-400 mesh; column 2X 10 cm) at a flow rate of 10 mllh. The column was washed with 50 ml 80 0/0 ethanol. Compounds responsible for cell division activity were eluted from the column with 250 ml 5 N NH 40H. The ammonia was removed under vacuum and the residue redissolved in 4 ml 80 Ofo ethanol. These Dowex purified extracts were loaded onto Whatman No. 1 chromatography paper and separated with iso-propanol: 25 Ofo ammonium hydroxide: water (10 : 1 : 1 v/v). The chromatograms were dried in a stream of air, divided into ten equal R f strips and these then assayed for cytokinin activity with the soybean callus bioassay. Intact bulbs and bulb scales were assayed on 30' ml of medium in 50 ml erlenmeyer flasks while root material was assayed on 15 ml medium in 25 ml culture flasks.
Results and Discussion Temperature had a pronounced effect on the growth and development of daffodil bulbs. Most rapid growth occurred at 10°C, where after 28 days, roots were well Z. Pflanzenphysiol. Bd. 86. S. 323-330. 1978.
Cytokinins in daffodil bulbs
325
developed irrespective of whether or not the bulbs were stored dry or moist. At 26 °C root development was slow (Fig. 1). After 56 days of storage the root systems and the shoots of bulbs stored at low temperature (10 °C) in moist vermiculite were well developed (Fig. 2). Where the bulbs were stored dry (10 °C) root development had not advanced further than was the case after 28 days of storage. Visible signs of shoot growth could however be observed. Bulbs stored moist at 26 °C for 56 days had well developed root systems and shoot growth was taking place. Those bulbs stored dry at the higher temperature (26 °C) did however, not show any visible signs of root development.
Fig. 1: The development of daffodil bulbs stored moist and dry at 10 °C and 26 °C for 28 days. A = 10 °C moist; B = 10 °C dry; C = 26 °C moist; D = 26 °C dry.
Fig. 2: The development of daffodil bulbs stored moist and dry at 10 °C and 26 °C for 56 days. A = 10 °C moi st ; B = 10 °C dry; C = 26 °C moist; D = 26 °C dry. Z. PJlanzenphysiol. Ed. 86. S. 323-330. 1978.
326
]. VAN
STADEN
Low levels of endogenous cytokinins were detected in the intact bulbs at the commencement of the experiment (Fig. 3). Two peaks of cytokinin activity were recorded after paper chromatography. The first was slow-moving and co-chromatographed with zeatin glucoside (Rf 0.2-0.5), while the second was fast-moving and co-chromatographed with zeatin and zeatin riboside (Rf 0.5-0.8). Both quantitative and qualitative changes in the endogenous cytokinins in daffodil bulbs were recorded during the course of storage. The most pronounced increase in cytokinin activity occurred in those bulbs stored at 10°C, After 56 days of storage the activity was highest in those bulbs stored moist (Fig. 4). The recorded increase in activity at low temperature was due to increases in both the slow- and fast-moving cytokinins (Fig. 3). In the bulbs stored in the cold under moist conditions the free base cytokinins, zeatin and zeatin riboside, were responsible for most of the recorded activity. Under dry conditions however, most activity was attributable to compounds that co-chromatographed with the cytokinin glucosides. These derivatives of zeatin and zeatin riboside are considered to be storage forms (PARKER and LETHAM, 1973) and apparently accumulate in plant tissues that are not rapidly growing (VAN STADEN and PAPAPHILIPPOU, 1977). Storage at 26°C had a less pronounced effect on the endogenous cytokinin levels in the bulbs. In the bulbs stored dry the total cytokinin content did not significantly change from that of the 26 C
0.4
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Fig. 3: The effect of moisture and temperature on the endogenous cytokinin levels of intact daffodil bulbs stored at 10°C and 26 °C for 28 and 56 days respectively. The equivalent of 50 g plant material was purified on Dowex 50 and the extracts chromatographed with iso-propanol: 25 % ammonium hydroxide: water (10 : 1 : 1 v/v). ZG = zeatin glucoside; ZR = zeatin riboside; Z = zeatin. Z. Pjlanzenphysiol. Bd. 86. S. 323-330. 1978.
Cytokinins in daffodil bulbs 10
327
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Fig. 4: Total callus yield of intact daffodil bulbs stored both moist and dry for 28 and 56 days at 10 DC and 26°C respectively. Values were obtained by adding the callus yield recorded after paper chromatography. A = 26 DC dry; B = 26 DC moist; C = 10 DC dry; D = 10 DC moist.
control bulbs. Under moist conditions there was, however, an increase in activity after 56 days of storage (Fig 4). This increase was mainly attributable to an increase in those compounds that co-chromatographed with zeatin and zeatin riboside (Fig. 3). From the above results it is noticeable that there appears to be a close correlation between the cytokinin content in the intact plants and the extent to which root development had occurred. As the roots are considered to be major sites of cytokinin synthesis (KENDE, 1964) it was decided to analyse roots and shoots separately in order to establish more closely the distribution of cytokinins within the bulbs. In bulbs stored dry and moist at 10°C most of the cytokinin activity was present in the roots that developed on the bulbs (Fig. 5). In the dry bulbs most of the activity co-chromatographed with zeatin glucoside while in the moist bulbs the major peak of activity chromatographed with zeatin and zeatin riboside. The presence of the storage cytokinins in the roots of dry stored bulbs is probably related to the fact that the endogenous cytokinins synthesized in the roots are not transported to the shoots. As a result of this they are inactivated in the roots where they accumulate. From the Z. Pflanzenphysiol. Bd. 86. S. 323-330. 1978.
328
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Fig. 5: The endogenous cytokinin levels in the roots and base plates and shoots of daffodil bulbs stored under moist and dry conditions for 56 days at 10 DC. Extracts were purified on Dowex 50 and the extracts chromatographed with iso-propanol: 25 % ammonium hydroxide: water (10 : 1 : 1 v/v). ZG = zeatin glucoside; ZR = zeatin riboside; Z = zeatin.
results it would appear that shoot development is largely dependent on the extent to which roots develop under the different storage conditions and on the synthesis and translocation of cytokinins from these organs. Gibberellins have been shown to increase in bulbs as a result of cold storage and it was suggested that roots may either be sites of synthesis or may provide the precursors for gibberellin production (REES, 1972). AUNG, DE HERTOGH and STABY (1971) have shown that endogenous gibberellin levels in bulbs are affected by moisture and temperature. The highest gibberellin content was recorded in bulbs kept moist at 9 DC. The removal of the base plates of the bulbs considerably reduced the gibberellin levels. From the present data it can be seen that the endogenous cytokinins were produced in the roots and base plates and were affected by moisture and temperature. In seeds, low temperatures have also been shown to increase endogenous cytokinin levels (VAN STADEN, WEBB and WAREING, 1972; BROWN and VAN STADEN, Z. Pjlanzenphysiol. Bd. 86. S. 323-330. 1978.
Cytokinins in daffodil bulbs
1973; DIMALLA and known at present.
VAN STADEN,
329
1977). How this increase is brought about is not
Acknowledgements The financial assistance of the Council for Scientific and Industrial Research, Pretoria, is gratefully acknowledged.
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