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Effect of Chilling Stratification on Nuclear Nucleic Acid Content in the Radicle of the Embryos of Tilia platyphyllos Scop
Short Communication
Effect of Chilling Stratification on Nuclear Nucleic Acid Content in the Radicle of the Embryos of Tilia platyphyllos Scop.
1) Department of Plant Physiology, AttilaJ6zsefUniversity, H-6701 Szeged, Hungary 2) Research Station of Research Institute for Fruit and Ornamental Plant Growing, H-4244 Ujfehen6, Hungary
3) Department of Botany, Attila J6zsef University, H-6701 Szeged, Hungary Received April 8, 1983 . Accepted July 18,1983
Summary The nucleic acid content in the radicle of embryos excised from chilled-stratified and treated at 25°C seeds was measured by a cytochemical method. No differences in the DNA contents of the nuclei were caused by either the inductive or non-inductive treatment. The RNA-content of the radicle of embryos excised from the chilled-stratified seeds was, however, higher than in embryos treated at 25°C for identical periods.
Key words: Tilia platyphyllos, germination, nucleic acid content, radicle.
Introduction The seeds of Tilia platypbyllos require chilling stratification for 150-180 days. During this period the physiological and biochemical changes in the seed leading to succesful germination take place. Successful germination denotes here not only the emerging of the radicle from the seed but also the development of the embryo to a normal seedling. Though the germination of treated at 25°C seeds can also be induced by mechanicaly removal of the endosperm tissues around the radicle, the capability of growth of the seedling obtained in this way is less than that of seedlings obtained from seeds chilled-stratified for an identical period (Nagy et at, 1981). Our results so far obtained concerning the dormancy of lime seeds indicate that one of the main roles of chilling stratification in the control of germination is the decrease of the mechanical resistance of the seed tissues, since the cytolysis of the endosperm tissues around the radicle can be observed only in chilled-stratified seeds (Nagy et al., Abbreviations: AU, arbitrary units; GA 3, gibberellic acid; GCA, gallocianine-chrome-alum; TCA, trichloroacetic acid. Z. Pjlanzenphysiol. Ed. 112. S. 187-189. 1983.
188
MARIA NAGY, TAMAs BUBAN and SZE1~EN PATAKY
1982). Due to the presence of hydrolases cytolysis of this type can occur even within the endosperm itself Oacobsen et aI., 1976) or due to the effect of hydrolases liberated from the embryo (Ikuma and Thimann, 1963; Berjak and Villiers, 1972; Junttila, 1973). In view of the role of nucleic acids in governing enzyme synthesis, our first step was the measurement of the content of nucleic acid in the radicle of embryos excised from chilled-stratified and treated at 25°C seeds, using a cytochemical method.
Materials and Methods Material was obtained from the Forestry of Csongrad County (Southern Hungary). After removal of the seed coats the seeds were disinfected with sodium hypochlorite and arranged in layers in sterile sand moistened to 70% of the full water capacity. After 5 months of chillingstratification at + 5°C and 5 months of treatment at 25°C the embryos were excised from the seeds and fixed in a 6: 1 : 1.3 mixture of ethanol: formaldehyde: picric acid (Halfacre et al., 1967), embedded in the usual way in paraffin, and longitudinal sections of a thickness of 6-8 /Lm prepared. For the sake of comparison embryos were also excised from seeds swollen for a week at 25°C. The DNA content of the cell nuclei was measured according to Heseman and Buban (1973). Hydrolysis was carried out in 1 N TCA at 25°C for 3 min, then at 50 °C for 15 min. Staining was carried out with Schiff reagent (HCI was substituted by TCA) at 25°C for 60 min. The content of DNA + RNA was measured according to Mitchell (1968) by staining with GCA. The site of measurements was the strip between two lines which can be drawn at a distance of about 400 and 500/Lm from the apex of the calyptra. The tissue regions investigated were: dermatogen, periblem, plerome. For the cytophotometric measurements an Amplival microscope (Zeiss) equipped with a measuring unit of MFV 4001 type was used. Objective: 63x, measure-diaphragm 0.75 and 0.5. Measurements were carried out at 550 and 525 nm respectively. Data have been expressed as arbitrary units (AU).
Results and Discussion Our results are summarized in Table 1. The data of our investigations indicate no significant differences between the DNA contents of the cell nuclei from various Table 1: Nucleic acid content of cell nuclei in the radicle cells of embryos of Tilia platyphyllos. Treatment at 25°C
Chilling stratification Tissue regIOns
Number of nuclei estimated
Arbitrary units
Number of nuclei estimated
Arbitrary units
DNA Dermatogen DNA+RNA
32 37
16.41 ± 1.06 38.16±2.27
28 34
16.02±2.74 27.25±2.46
Periblem
DNA DNA + RNA
29 40
19.07 ± 1.02 36.61±2.98
39 43
19.86±2.57 25.30± 1.94
Plerome
DNA DNA + RNA
35 30
16.00± 1.07 31.80±2.23
48 36
15.43±2.06 22.60±2.92
Estimation of nuclei content of
Z. Pjlanzenphysiol. Bd. 112. S. 187-189. 1983.
Nucleic acid content in Tilia embryo radicles
189
tissue regions of the treated at 25°C and of the chilled-stratified embryos. Consequently, the physiological activity of lime seeds cannot be evaluated on the basis of the DNA levels, in complete contrast to the seeds of Agrostemma githago (Hecker and Maibauer, 1976) and of Haplopappus gracilis (Galli et aI., 1981). On the basis of our data concerning the contents of DNA + RNA, the RNA contents of the cell nuclei in the cells of embryos chilled-stratified were in all tissue regions investigated significantly (P = 0.01 level of error) higher than those in the embryos treated at 25°C for an identical period. In the cells of embryos excised from seeds swollen for a week at 25 °C the content of nucleic acid of seeds did not differ from those of the embryos treated at 25°C for 5 months. Villiers (1968) also reported the inhibition of the RNA synthesis in the embryos in dormant seeds of Fraxinus excelsior, which are in many respects similar to the seeds of lime. Similar data have been published by Khan (1970) for the embryos of the nonstratified Pyrus communis var. Bartlett. These data point to a similar mechanism of inhibition. The embryos of lime seeds grow during treatment at 25°C (Nagy et aI., 1981). Their growth can be promoted within the seeds by exogenous GA3 treatment (Nagy et al., 1982) but, even so, emergence of the radicle occurs in this latter case only when the treatment is combined with chilling-stratification. Thus, chilling-stratification may play a role in starting the synthesis of enzymes which make possible the emergence of the radicle i.e. in suspending the inhibition of the synthesis of m-RNA's of a certain type. References BERJAK, P. and T. A. VILLIERS: New Phytol. 71,135-144 (1972). GALLI, M. G., M. LEVI, and E. SPARVOU: Physiol. Plant. 51, 321-325 (1981). HALFACRE, R. G., A. M. WALTON, andJ. C. OSBORNE: Hort. Sci. 2, 116 (1967). HECKER, M. and B. MAIBAUER: Biochem. Physiol. Pflanzen 170, 363-379 (1976). HESEMANN, C. U. and T. BUBAN: Histochemie, 36, 237-246 (1973). IKUMA, H. and K. V. THIMAN: Plant Cell Physiol. 4, 169-185 (1963). JACOBSEN, J. V., E. PRESSMAN, and N. A. PYLIOTlS: Planta, 129, 113-122 (1976). JUNTTILA, 0.: Physiol. Plant. 29, 256-263 (1973). KHAN, A. A.: In: Plant Growth Substances, 205-215. Springer-Verlag, New York, 1970. MITCHELL, J.: Histochem. J. 1, 106-123 (1968). NAGY, M., S. PATAKY and A. KERI: Acta BioI. Szeged, 27,127-137 (1981). NAGY, M., S. PATAKY and A. KERI: Acta BioI. Szeged, 28,11-117 (1982). VILUERS, T. A.: Planta, 82, 342-354 (1968).
Z. Pjlanzenphysiol. Bd. 112. S. 187-189. 1983.
Effect of Chilling Stratification on Nuclear Nucleic Acid Content in the Radicle of the Embryos of Tilia platyphyllos Scop.
1) Department of Plant Physiology, AttilaJ6zsefUniversity, H-6701 Szeged, Hungary 2) Research Station of Research Institute for Fruit and Ornamental Plant Growing, H-4244 Ujfehen6, Hungary
3) Department of Botany, Attila J6zsef University, H-6701 Szeged, Hungary Received April 8, 1983 . Accepted July 18,1983
Summary The nucleic acid content in the radicle of embryos excised from chilled-stratified and treated at 25°C seeds was measured by a cytochemical method. No differences in the DNA contents of the nuclei were caused by either the inductive or non-inductive treatment. The RNA-content of the radicle of embryos excised from the chilled-stratified seeds was, however, higher than in embryos treated at 25°C for identical periods.
Key words: Tilia platyphyllos, germination, nucleic acid content, radicle.
Introduction The seeds of Tilia platypbyllos require chilling stratification for 150-180 days. During this period the physiological and biochemical changes in the seed leading to succesful germination take place. Successful germination denotes here not only the emerging of the radicle from the seed but also the development of the embryo to a normal seedling. Though the germination of treated at 25°C seeds can also be induced by mechanicaly removal of the endosperm tissues around the radicle, the capability of growth of the seedling obtained in this way is less than that of seedlings obtained from seeds chilled-stratified for an identical period (Nagy et at, 1981). Our results so far obtained concerning the dormancy of lime seeds indicate that one of the main roles of chilling stratification in the control of germination is the decrease of the mechanical resistance of the seed tissues, since the cytolysis of the endosperm tissues around the radicle can be observed only in chilled-stratified seeds (Nagy et al., Abbreviations: AU, arbitrary units; GA 3, gibberellic acid; GCA, gallocianine-chrome-alum; TCA, trichloroacetic acid. Z. Pjlanzenphysiol. Ed. 112. S. 187-189. 1983.
188
MARIA NAGY, TAMAs BUBAN and SZE1~EN PATAKY
1982). Due to the presence of hydrolases cytolysis of this type can occur even within the endosperm itself Oacobsen et aI., 1976) or due to the effect of hydrolases liberated from the embryo (Ikuma and Thimann, 1963; Berjak and Villiers, 1972; Junttila, 1973). In view of the role of nucleic acids in governing enzyme synthesis, our first step was the measurement of the content of nucleic acid in the radicle of embryos excised from chilled-stratified and treated at 25°C seeds, using a cytochemical method.
Materials and Methods Material was obtained from the Forestry of Csongrad County (Southern Hungary). After removal of the seed coats the seeds were disinfected with sodium hypochlorite and arranged in layers in sterile sand moistened to 70% of the full water capacity. After 5 months of chillingstratification at + 5°C and 5 months of treatment at 25°C the embryos were excised from the seeds and fixed in a 6: 1 : 1.3 mixture of ethanol: formaldehyde: picric acid (Halfacre et al., 1967), embedded in the usual way in paraffin, and longitudinal sections of a thickness of 6-8 /Lm prepared. For the sake of comparison embryos were also excised from seeds swollen for a week at 25°C. The DNA content of the cell nuclei was measured according to Heseman and Buban (1973). Hydrolysis was carried out in 1 N TCA at 25°C for 3 min, then at 50 °C for 15 min. Staining was carried out with Schiff reagent (HCI was substituted by TCA) at 25°C for 60 min. The content of DNA + RNA was measured according to Mitchell (1968) by staining with GCA. The site of measurements was the strip between two lines which can be drawn at a distance of about 400 and 500/Lm from the apex of the calyptra. The tissue regions investigated were: dermatogen, periblem, plerome. For the cytophotometric measurements an Amplival microscope (Zeiss) equipped with a measuring unit of MFV 4001 type was used. Objective: 63x, measure-diaphragm 0.75 and 0.5. Measurements were carried out at 550 and 525 nm respectively. Data have been expressed as arbitrary units (AU).
Results and Discussion Our results are summarized in Table 1. The data of our investigations indicate no significant differences between the DNA contents of the cell nuclei from various Table 1: Nucleic acid content of cell nuclei in the radicle cells of embryos of Tilia platyphyllos. Treatment at 25°C
Chilling stratification Tissue regIOns
Number of nuclei estimated
Arbitrary units
Number of nuclei estimated
Arbitrary units
DNA Dermatogen DNA+RNA
32 37
16.41 ± 1.06 38.16±2.27
28 34
16.02±2.74 27.25±2.46
Periblem
DNA DNA + RNA
29 40
19.07 ± 1.02 36.61±2.98
39 43
19.86±2.57 25.30± 1.94
Plerome
DNA DNA + RNA
35 30
16.00± 1.07 31.80±2.23
48 36
15.43±2.06 22.60±2.92
Estimation of nuclei content of
Z. Pjlanzenphysiol. Bd. 112. S. 187-189. 1983.
Nucleic acid content in Tilia embryo radicles
189
tissue regions of the treated at 25°C and of the chilled-stratified embryos. Consequently, the physiological activity of lime seeds cannot be evaluated on the basis of the DNA levels, in complete contrast to the seeds of Agrostemma githago (Hecker and Maibauer, 1976) and of Haplopappus gracilis (Galli et aI., 1981). On the basis of our data concerning the contents of DNA + RNA, the RNA contents of the cell nuclei in the cells of embryos chilled-stratified were in all tissue regions investigated significantly (P = 0.01 level of error) higher than those in the embryos treated at 25°C for an identical period. In the cells of embryos excised from seeds swollen for a week at 25 °C the content of nucleic acid of seeds did not differ from those of the embryos treated at 25°C for 5 months. Villiers (1968) also reported the inhibition of the RNA synthesis in the embryos in dormant seeds of Fraxinus excelsior, which are in many respects similar to the seeds of lime. Similar data have been published by Khan (1970) for the embryos of the nonstratified Pyrus communis var. Bartlett. These data point to a similar mechanism of inhibition. The embryos of lime seeds grow during treatment at 25°C (Nagy et aI., 1981). Their growth can be promoted within the seeds by exogenous GA3 treatment (Nagy et al., 1982) but, even so, emergence of the radicle occurs in this latter case only when the treatment is combined with chilling-stratification. Thus, chilling-stratification may play a role in starting the synthesis of enzymes which make possible the emergence of the radicle i.e. in suspending the inhibition of the synthesis of m-RNA's of a certain type. References BERJAK, P. and T. A. VILLIERS: New Phytol. 71,135-144 (1972). GALLI, M. G., M. LEVI, and E. SPARVOU: Physiol. Plant. 51, 321-325 (1981). HALFACRE, R. G., A. M. WALTON, andJ. C. OSBORNE: Hort. Sci. 2, 116 (1967). HECKER, M. and B. MAIBAUER: Biochem. Physiol. Pflanzen 170, 363-379 (1976). HESEMANN, C. U. and T. BUBAN: Histochemie, 36, 237-246 (1973). IKUMA, H. and K. V. THIMAN: Plant Cell Physiol. 4, 169-185 (1963). JACOBSEN, J. V., E. PRESSMAN, and N. A. PYLIOTlS: Planta, 129, 113-122 (1976). JUNTTILA, 0.: Physiol. Plant. 29, 256-263 (1973). KHAN, A. A.: In: Plant Growth Substances, 205-215. Springer-Verlag, New York, 1970. MITCHELL, J.: Histochem. J. 1, 106-123 (1968). NAGY, M., S. PATAKY and A. KERI: Acta BioI. Szeged, 27,127-137 (1981). NAGY, M., S. PATAKY and A. KERI: Acta BioI. Szeged, 28,11-117 (1982). VILUERS, T. A.: Planta, 82, 342-354 (1968).
Z. Pjlanzenphysiol. Bd. 112. S. 187-189. 1983.