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Surface morphology of narcissus flowering stems as revealed by scanning electron microscopy
Micron, 1978, Vol. : 9, pp. 95-97.
0047-7206[78/0601-0095
$02.00/0
© Pergamon Press Ltd. Printed in Great Britain
SHORT COMMUNICATION SURFACE
MORPHOLOGY
AS REVEALED
BY
OF
NARCISSUS
SCANNING
FLOWERING
ELECTRON
STEMS
MICROSCOPY
D. JONES Department of Microbiology, The Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen AB9 2QJ, Scotland (Received 27 July 1977; received for publication 20 January 1978)
Al~traet--Morphological features of narcissus flower stem surfaces in a scanning electron microscope varied according to the preparative techniques used. Thus epi-cuticular wax configurations were seen on unfixed and prefixed flowering stems, which had been freeze-dried,but not on those which were critical point dried.
The dried plant segments were coated with The introduction of the scanning electron microscope has enabled significant improve- carbon followed by gold/palladium alloy (Au, ments to be made in our knowledge of the sur- 81.5 %, Pd, 18.5 %) and examined in a Cambridge face features of microbiological and botanical Stereoscan $4 SEM operated at 30kV. Specispecimens. However, the preparation of the mens were usually tilted 45 ° to the main beam. Flower-stem wax is readily identified on freezespecimens prior to their examination in the microscope needs special care (Parsons et al., dried material (Figs. 1 and 2) whereas it is 1974). The present communication illustrates this obviously removed by critical-point drying (Figs. point by comparing two techniques commonly 3 and 4). It had been noted previously that wax used to dehydrate specimens. The observations is similarly removed from pea leaf surfaces reported here were made during an investigation (Jones, 1976). The present communication on scape (flowering stem) and leaf wax of shows that much of the wax on the narcissus narcissus (cultivar Golden Harvest). This wax stems can be removed either during dehydration was sometimes altered where the plants had in the alcohol series (Figs. 5 and 6) or by steeping been invaded by pathogenic organisms and the the plant material in Freon 113 over night (Figs. detailed results of these infection studies will be 7 and 8). What remains of the wax on alcoholreported elsewhere. extracted stems is somewhat different in strucPortions of plant material were either frozen ture from that seen on freeze-dried material. rapidly in trays floated on liquid nitrogen and Wax was evident on flowering stems which were dried in a Pearse tissue drier overnight (Jones, air-dried with no prior treatment but such 1976) or critical-point dried by the following samples proved unstable to the electron beam, procedure. Portions were dehydrated in an ethyl although they were coated with metal in the alcohol series (70~o, 90%, 100%) and then usual way. The wax was not removed from stem passed through a series of Freon 113/ethyl alco- surfaces which had been pre-fixed in 3% hol mixtures of the following proportions; 1 : 4, glutaraldehyde in phosphate buffer at pH 6.8, 2: 3, 3: 2, 4: 1 and finally through Freon 113 (Du followed by 2 % osmic acid in the same buffer, Pont) alone. They were then critical-point dried and then freeze-dried. However, wax was refrom Freon 13 (Imperial Chemical Industries . moved from stem surfaces similarly pre-fixed Ltd) or carbon dioxide. ~ b u t subsequently critical-point dried. 95
96
D. Jones
F r o m the data given, the particular preparative technique chosen should obviously depend on the ultrastructural features under investigation. E x a m i n a t i o n of specimens in the frozen state (Echlin and Moreton, 1973) could minimize the artefacts introduced by any particular technique employed. Acknowledoements--I am grateful to Dr. Elizabeth Gray of the Plant Pathology Division, North of Scotland College of Agriculture for providing the plant material. Miss Helen Geddes provided skilled aid in preparing the material for examination in the electron microscope.
REFERENCES Echlin, P. and Moreton, R., 1973. The preparation, coating and examination of frozen biological materials in the SEM. In: Scanning Electron Microscopy, 1973 (Part IlI). Proceedings of the workshop on Scanning Electron Microscopy in Pathology. lIT Research Institute, Chicago, Illinois, U.S.A., pp. 326-332. Jones, D., 1976. Infection of plant tissue by Sclerotinia sclerotiorum: a scanning electron microscope study. Micron, 7: 275-279. Parsons, E., Bole, B., Hall, D. J. and Thomas, W. D. E., 1974. A comparative survey of techniques for preparing plant surfaces for the scanning electron microscope. J. Microsc., 101: 59-75.
Legend for Page 97 Figs. 1-8. Effect of different preparative techniques on epi-cuticular wax of narcissus flowering stems, as revealed by scanning electron microscopy. Figs. 1. and 2. Wax on freeze-dried material. Fig. 1, × 900; Fig. 2, x 3750. Figs. 3 and 4. Wax-free surfaces of critical-point dried material. Fig. 3, × 350; Fig. 4, x 1500. Figs. 5 and 6. Material dehydrated in ethyl alcohol series (70 %, 90 %, 100 %). Fig. 5, × 380; Fig. 6, × 1350. Figs. 7 and 8. Material steeped in Freon 113 overnight, then air dried. Fig. 7, ×400; Fig. 8, 1400.
SEM of Narcissus Flowering Stem
97
0047-7206[78/0601-0095
$02.00/0
© Pergamon Press Ltd. Printed in Great Britain
SHORT COMMUNICATION SURFACE
MORPHOLOGY
AS REVEALED
BY
OF
NARCISSUS
SCANNING
FLOWERING
ELECTRON
STEMS
MICROSCOPY
D. JONES Department of Microbiology, The Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen AB9 2QJ, Scotland (Received 27 July 1977; received for publication 20 January 1978)
Al~traet--Morphological features of narcissus flower stem surfaces in a scanning electron microscope varied according to the preparative techniques used. Thus epi-cuticular wax configurations were seen on unfixed and prefixed flowering stems, which had been freeze-dried,but not on those which were critical point dried.
The dried plant segments were coated with The introduction of the scanning electron microscope has enabled significant improve- carbon followed by gold/palladium alloy (Au, ments to be made in our knowledge of the sur- 81.5 %, Pd, 18.5 %) and examined in a Cambridge face features of microbiological and botanical Stereoscan $4 SEM operated at 30kV. Specispecimens. However, the preparation of the mens were usually tilted 45 ° to the main beam. Flower-stem wax is readily identified on freezespecimens prior to their examination in the microscope needs special care (Parsons et al., dried material (Figs. 1 and 2) whereas it is 1974). The present communication illustrates this obviously removed by critical-point drying (Figs. point by comparing two techniques commonly 3 and 4). It had been noted previously that wax used to dehydrate specimens. The observations is similarly removed from pea leaf surfaces reported here were made during an investigation (Jones, 1976). The present communication on scape (flowering stem) and leaf wax of shows that much of the wax on the narcissus narcissus (cultivar Golden Harvest). This wax stems can be removed either during dehydration was sometimes altered where the plants had in the alcohol series (Figs. 5 and 6) or by steeping been invaded by pathogenic organisms and the the plant material in Freon 113 over night (Figs. detailed results of these infection studies will be 7 and 8). What remains of the wax on alcoholreported elsewhere. extracted stems is somewhat different in strucPortions of plant material were either frozen ture from that seen on freeze-dried material. rapidly in trays floated on liquid nitrogen and Wax was evident on flowering stems which were dried in a Pearse tissue drier overnight (Jones, air-dried with no prior treatment but such 1976) or critical-point dried by the following samples proved unstable to the electron beam, procedure. Portions were dehydrated in an ethyl although they were coated with metal in the alcohol series (70~o, 90%, 100%) and then usual way. The wax was not removed from stem passed through a series of Freon 113/ethyl alco- surfaces which had been pre-fixed in 3% hol mixtures of the following proportions; 1 : 4, glutaraldehyde in phosphate buffer at pH 6.8, 2: 3, 3: 2, 4: 1 and finally through Freon 113 (Du followed by 2 % osmic acid in the same buffer, Pont) alone. They were then critical-point dried and then freeze-dried. However, wax was refrom Freon 13 (Imperial Chemical Industries . moved from stem surfaces similarly pre-fixed Ltd) or carbon dioxide. ~ b u t subsequently critical-point dried. 95
96
D. Jones
F r o m the data given, the particular preparative technique chosen should obviously depend on the ultrastructural features under investigation. E x a m i n a t i o n of specimens in the frozen state (Echlin and Moreton, 1973) could minimize the artefacts introduced by any particular technique employed. Acknowledoements--I am grateful to Dr. Elizabeth Gray of the Plant Pathology Division, North of Scotland College of Agriculture for providing the plant material. Miss Helen Geddes provided skilled aid in preparing the material for examination in the electron microscope.
REFERENCES Echlin, P. and Moreton, R., 1973. The preparation, coating and examination of frozen biological materials in the SEM. In: Scanning Electron Microscopy, 1973 (Part IlI). Proceedings of the workshop on Scanning Electron Microscopy in Pathology. lIT Research Institute, Chicago, Illinois, U.S.A., pp. 326-332. Jones, D., 1976. Infection of plant tissue by Sclerotinia sclerotiorum: a scanning electron microscope study. Micron, 7: 275-279. Parsons, E., Bole, B., Hall, D. J. and Thomas, W. D. E., 1974. A comparative survey of techniques for preparing plant surfaces for the scanning electron microscope. J. Microsc., 101: 59-75.
Legend for Page 97 Figs. 1-8. Effect of different preparative techniques on epi-cuticular wax of narcissus flowering stems, as revealed by scanning electron microscopy. Figs. 1. and 2. Wax on freeze-dried material. Fig. 1, × 900; Fig. 2, x 3750. Figs. 3 and 4. Wax-free surfaces of critical-point dried material. Fig. 3, × 350; Fig. 4, x 1500. Figs. 5 and 6. Material dehydrated in ethyl alcohol series (70 %, 90 %, 100 %). Fig. 5, × 380; Fig. 6, × 1350. Figs. 7 and 8. Material steeped in Freon 113 overnight, then air dried. Fig. 7, ×400; Fig. 8, 1400.
SEM of Narcissus Flowering Stem
97